Identification and Characterization of the Pheromones in the Minute Pirate Bug Orius sauteri (Heteroptera: Anthocoridae).

The flower bug Orius sauteri is a generalist predator that occurs throughout Japan, and is a promising indigenous natural enemy for micro-pests such as thrips, aphids, and spider mites. We aimed to manipulate the attraction, dispersal, and settlement behavior of Orius bugs using natural chemical substances emitted by the bugs themselves. To identify potential candidates, we screened components in the whole-body extract of O. sauteri based on antennal response and then determined their chemical structure. A gas chromatograph electroantennographic detector (GC/EAD) indicated that the antennae of males responded to two components in the extract of females. GC/mass spectrometry (MS) showed that these two components were octenal and octadienal. Derivatization or GC-FT-IR analysis identified these components as (E)-2-octenal and (E)-2,7-octadienal. To assess the effect of these components on O. sauteri behavior, we conducted two assays. A field bioassay demonstrated that a blend of the two components functioned as a sex pheromone, and a dispersal assay showed that (E)-2-octenal generated a dose-dependent dispersal response. Our study will provide baseline information for enhancing the retention of O. sauteri on important commercial crops to prey on pest species.

Contact Sex Pheromone Activity of Synthetic Gomadalactones in Male White-Spotted Longhorn Beetle, Anoplophora malasiaca (Coleoptera: Cerambycidae).

The elytra of females of the white-spotted longhorn beetle, Anoplophora malasiaca (Coleoptera: Cerambycidae), are coated with a contact sex pheromone, which was previously shown to be composed of at least three chemical groups. Individually, the chemical groups had little pheromonal activity, but a blend of all three exhibited activity equal to that of the crude female extract. Two groups are female-specific aliphatic hydrocarbons and ketones, which were previously synthesized and confirmed to elicit mating behavior. The third group consists of three lactones, gomadalactones A, B, and C, whose chemical structures were previously identified. These have now been synthesized, and the contact sex pheromone activities of synthetic gomadalactones A, B, and C, and the diastereomer of gomadalactone C, were tested in bioassays in this study. When tested in combination with synthetic hydrocarbons and ketones at the same doses as found in female elytra extract, the individual gomadalactones and a blend showed potent pheromonal activity equivalent to that of the crude extract of the elytra of female beetles. This completes the identification of the essential components of the contact sex pheromone of A. malasiaca. Redundancy of components in the hydrocarbon and ketone groups required to elicit mating behavior was observed previously, and this was also true for the gomadalactones.

Female Sex Pheromone in Trails of the Minute Pirate Bug, Orius minutus (L).

Orius minutus (L.) (Heteroptera: Anthocoridae) is a natural enemy of agricultural pests such as thrips, aphids, and various newly hatched insect juveniles. In this study, we conducted 1) behavioral assays for evidence of contact sex pheromone activity in trails of O. minutus, and 2) chemical analysis to identify the essential chemical components of the trails. Males showed arrestment to trails of mature virgin females but not to trails from either conspecific nymphs or immature females. Females also showed arrestment to trails from conspecific males, although the response was weaker than that exhibited by males. The activity of female trails lasted for at least 46 h after deposition. Males showed a response irrespective of mating experience. Following confirmation that a contact sex pheromone was present in the trails of female O. minutus, we used a bioassay-driven approach to isolate the active chemicals. After fractionation on silica gel, the n-hexane fraction was found to be biologically active to males. A major compound in the active fraction was (Z)-9-nonacosene; this compound was found only in trail extracts of mature virgin females. Synthetic (Z)-9-nonacosene arrested O. minutus males, indicating that it is the major active component of the contact sex pheromone in the trails of female O. minutus.

Chemical and Physical Cues Synergistically Affect Mating Behavior Sequences of Male Dasylepida ishigakiensis (Coleoptera: Scarabaeidae).

We investigated physical and chemical cues involved in male mating behavior of the white grub beetle, Dasylepida ishigakiensis (Scarabaeidae). When presented with female attractant pheromone (R)-2-butanol lures in a flight tunnel, nearly all males exhibited orientation and touching behaviors to freshly killed males and females and to intact glass models. Males landed and bent their abdomens on male and female bodies, but not on intact glass models. When treated with one female equivalent (FE) extract, washed immature male bodies and glass models both evoked stronger male responses than untreated equivalents, with the former eliciting a greater response than the treated glass models. Male responses to target male and female bodies decreased with increased numbers of washings of target bodies with organic solvents. These results suggest that the chemical factors that elicit male abdominal bending behavior are present on the body surface in both sexes. Washed immature male bodies treated with 1 FE or one male equivalent (ME) of extract induced strong male abdominal bending behavior. Washed mature female bodies treated with 1 ME extract also evoked male responses. Extracts of both sexes included factors eliciting male abdominal bending behavior. These results suggest that both physical and chemical cues derived from conspecifics cooperate to facilitate male mating recognition in D. ishigakiensis. The mating process of this species in the field is highly synchronized. Thus, after orienting to a female-like object, the only information males require by touching is whether the sex attractant pheromone that attracted them is indeed from a conspecific.

Anoplophora glabripennis, an invasive longhorned beetle, has the potential to damage fruit trees in Japan.

Invasive Anoplophora glabripennis recently became established in Japan and has caused heavy damage to several street-tree species. Overseas, A. glabripennis infests trees of the genera Acer and Populus as common host plants, and Malus, Pyrus, and Prunus (Rosaceae), including apple, pear, and plum trees; it therefore poses a potential risk to the production of economically valuable fruits in Japan. Fruit farms in areas already invaded by A. glabripennis are now threatened with tree infestation. We aimed to determine the potential damage to major fruit species in Japan. In the laboratory, we determined if the adult beetle is attracted to the odor of each of these tree species' branches; two confirmed host plant species and five Rosaceae fruit species, as well as its feeding preferences among branches of one host plant and the five fruit trees and its oviposition preferences among them. Among the fruit species, cherry branch had the highest rate of odor orientation by males. The feeding-preference assay showed that, besides the host plant, Japanese pear was the most consumed among the fruit trees. The potential risk of A. glabripennis laying eggs on fruit-tree branches was high for Japanese pear and above zero for plum, apple, and cherry branches.

Detection of invasive and native beetle species within trees by chemical analysis of frass.

In recent years, several invasive woodborers (Coleoptera: Cerambycidae) have been found in Japan. Aromia bungii is a worldwide important pest of fruits and ornamental species of the genus Prunus. It invaded Japan in the early 2010s and now causes heavy damage to stone fruit trees. Anoplophora glabripennis and Apriona swainsoni are destructive pests of street, ornamental and horticultural trees. The first step in intercepting these beetles is to detect their presence early in their infestation, as accurate identification is crucial for their management. Ejected frass is a major sign of infestation and likely holds information on the insect. We focused on chemicals in both larvae and frass, and conducted a GC-MS analysis of these three invasive beetles and the native Anoplophora malasiaca. In all four species, 4 or 5 species-specific hydrocarbons were detected in both larvae and frass. These results indicate that analysis of hydrocarbons in frass could allow definitive detection of invasive wood-boring pests.

Differences in Male Mate Recognition between the Invasive Anoplophora glabripennis (Coleoptera: Cerambycidae) and Japanese Native A. malasiaca.

The Asian longicorn beetle Anoplophora glabripennis is a recently arrived invasive species to Japan. The Japanese native A. malasiaca shows an extensive overlap with A. glabripennis with host plants, niches, and emergence season. Hybridization between these two species is suspected in Japan. The surface of the female is covered with contact sex pheromones that elicit male mating behavior within species. We evaluated the contact pheromonal activity of crude extract and fractions of female A. glabripennis coated on a black glass model and revealed a hydrocarbon fraction and a blend of fractions to show activity but relatively weak, suggesting the presence of other unknown active compounds. Few male A. glabripennis showed mating behavior when they were exposed to a crude extract of female A. malasiaca. However, a considerable number of A. malasiaca males mounted and showed abdominal bending behavior when presented with glass models that were coated with each extract of female A. glabripennis and A. malasiaca. Gomadalactones are essential contact pheromone components that elicit mating behavior in male A. malasiaca; however, we could not detect them in female A. glabripennis extract. Here, we investigated the possible reasons for this phenomenon and the difference in male mate recognition systems between these two species.

Musty odor of entomopathogens enhances disease-prevention behaviors in the termite Coptotermes formosanus.

Termites often eliminate pathogens directly through mutual grooming, and are thereby prevent infections from entomopathogenic fungi. Our previous study confirmed that the antennae of Coptotermesformosanus sensitively responded to the musty odor of entomopathogenic fungi. However, it is unclear if this odor has any effect on termite behavior. The purpose of this study was to clarify the effects of fungal odor on termite behavior, especially on conidia removal. The musty odor was prepared as an aqueous solution by immersing conidia in distilled water. When untreated termites were mixed with fungal-odor-treated termites at a ratio of 4:1, mutual grooming and attack of treated termites were frequently observed. This indicated that the fungal odor triggered these behavioral responses. While some components of the fungal odor were found in all of the entomopathogenic fungi tested, the odor profiles differed among the isolates.

Behavioral changes in the termite, Coptotermes formosanus (Isoptera), inoculated with six fungal isolates.

The studies of pathogen-prevention behaviors of termites have focused on hygiene behavior directed only against highly virulent pathogens. Therefore, we compared behavioral changes in the subterranean termite Coptotermes formosanus following contact with entomopathogenic fungi with different levels of virulence. The fungal virulence was inferred from the daily mortality and the LD50 value in previous data. When untreated termites were allowed to contact their fungus-inoculated nestmates, mutual grooming was frequent during 30 min after inoculation. The inoculated termites were often attacked and eaten by their uninoculated nestmates, and then buried after death. Notably, there was no influence of fungal virulence on these pathogen-prevention behaviors. However, the fungal isolates and genera affected not only the frequency of the behaviors but also the horizontal transmission pattern, the number of dead individuals and the survival period before the first death following infection.

Improving Contagion and Horizontal Transmission of Entomopathogenic Fungi by the White-Spotted Longicorn Beetle, Anoplophora malasiaca, with Help of Contact Sex Pheromone.

The white-spotted longicorn beetle, Anoplophora malasiaca, is one of the most destructive pests of horticultural crops and street trees. Effective controls are needed because the effect of marketed insecticides is limited. Entomopathogenic fungi offer a solution, and improving the rate of infection would be a breakthrough in this beetle's control. The combination of pathogenic fungi and the beetle's contact sex pheromone was suggested. The surface of the female body is covered with contact sex pheromone, which elicit male mating behavior. To develop a method for the practical control of this beetle, we evaluated the arrestant activity of female extract containing contact pheromone coated on a black glass model. Males presented with a coated model held on for 5 h (mean) during an 8-h experiment. In contrast, males presented with a control model held on for <0.3 h. Males that held onto coated models attached to fabric impregnated with conidia of the fungus Beauveria brongniartii picked up much conidia, which they then passed on to females during mating.

Identification of a male-produced sex-aggregation pheromone for a highly invasive cerambycid beetle, Aromia bungii.

The longhorned beetle Aromia bungii (Coleoptera: Cerambycidae) is a major pest of stone fruit trees in the genus Prunus, including cherries, apricots, and peaches. Its native range includes China, Korea, Mongolia, and eastern Russia, but it has recently invaded and become established in several countries in Europe, and Japan, and it has been intercepted in shipments coming into North America and Australia. Here, we report the identification of its male-produced aggregation pheromone as the novel compound (E)-2-cis-6,7-epoxynonenal. In field trials in its native range in China, and in recently invaded areas of Japan, the pheromone attracted both sexes of the beetle. Thus, the pheromone should find immediate use in worldwide quarantine surveillance efforts to detect the beetle in incoming shipments. The pheromone will also be a crucial tool in ongoing efforts to eradicate the beetle from regions of the world that it has already invaded.

Behavioral responses to the alarm pheromone of the ant Camponotus obscuripes (Hymenoptera: Formicidae).

The alarm pheromone of the ant Camponotus obscuripes (Formicinae) was identified and quantified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Comparisons between alarm pheromone components and extracts from the major exocrine gland of this ant species revealed that the sources of its alarm pheromone are Dufour's gland and the poison gland. Most components of Dufour's gland were saturated hydrocarbons. n-Undecane comprised more than 90% of all components and in a single Dufour's gland amounted to 19 microg. n-Decane and n-pentadecane were also included in the Dufour's gland secretion. Only formic acid was detected in the poison gland, in amounts ranging from 0.049 to 0.91 microl. This ant species releases a mixture of these substances, each of which has a different volatility and function. When the ants sensed formic acid, they eluded the source of the odor; however, they aggressively approached odors of n-undecane and n-decane, which are highly volatile. In contrast, n-pentadecane, which has the lowest volatility among the identified compounds, was shown to calm the ants. The volatilities of the alarm pheromone components were closely related to their roles in alarm communication. Highly volatile components vaporized rapidly and spread widely, and induced drastic reactions among the ants. As these components became diluted, the less volatile components calmed the excited ants. How the worker ants utilize this alarm communication system for efficient deployment of their nestmates in colony defense is also discussed herein.

Host plant affects the sexual attractiveness of the female white-spotted longicorn beetle, Anoplophora malasiaca.

Anoplophora malasiaca (Coleoptera: Cerambycidae) is a serious pest that destroys various landscape and crop trees in Japan. We evaluated the precopulatory responses of three different A. malasiaca populations collected from mandarin orange, willow and blueberry trees. Most of the males accepted mates from within the same host plant population as well as females from the willow and blueberry populations. However, significant number of males from the blueberry and willow populations rejected females from the mandarin orange population immediately after touching them with their antennae. Because all three of the female populations produced contact sex pheromones on their elytra, the females of the mandarin orange population were predicted to possess extra chemicals that repelled the males of the other two populations. ß-Elemene was identified as a key component that was only found in mandarin orange-fed females and induced a rejection response in willow-fed males. Our results represent the first example of a female-acquired repellent against conspecific males of different host plant populations, indicating that the host plant greatly affects the female's sexual attractiveness.

Odor aversion and pathogen-removal efficiency in grooming behavior of the termite Coptotermes formosanus.

The results of biocontrol with entomopathogens in termites have been discouraging because of the strong social hygiene behavior for removing pathogens from termite colonies. However, the mechanism of pathogen detection is still unclear. For the successful application of biopesticides to termites in nature, it would be beneficial to identify substances that could disrupt the termite's ability to perceive pathogens. We hypothesized that termites can perceive pathogens and this ability plays an important role in effective hygiene behavior. In this study, pathogen-detection in the subterranean termite Coptotermes formosanus was investigated. We performed quantitative assays on conidia removal by grooming behavior using epifluoresence microscopy and Y-maze tests to examine the perception of fungal odor by termites. Three species each of high- and low-virulence entomopathogenic fungi were used in each test. The results demonstrated that termites removed conidia more effectively from a nestmate's cuticle if its odor elicited stronger aversion. Highly virulent pathogens showed higher attachment rates to termite surfaces and their odors were more strongly avoided than those of low-virulence isolates in the same species. Moreover, termites appeared to groom each other more persistently when they had more conidia on their bodies. In brief, insect perception of pathogen-related odor seems to play a role in the mechanism of their hygiene behavior.

Cuticular Hydrocarbon Content that Affects Male Mate Preference of Drosophila melanogaster from West Africa.

Intraspecific variation in mating signals and preferences can be a potential source of incipient speciation. Variable crossability between Drosophila melanogaster and D. simulans among different strains suggested the abundance of such variations. A particular focus on one combination of D. melanogaster strains, TW1(G23) and Mel6(G59), that showed different crossabilities to D. simulans, revealed that the mating between females from the former and males from the latter occurs at low frequency. The cuticular hydrocarbon transfer experiment indicated that cuticular hydrocarbons of TW1 females have an inhibitory effect on courtship by Mel6 males. A candidate component, a C25 diene, was inferred from the gas chromatography analyses. The intensity of male refusal of TW1 females was variable among different strains of D. melanogaster, which suggested the presence of variation in sensitivity to different chemicals on the cuticle. Such variation could be a potential factor for the establishment of premating isolation under some conditions.

Chemical discrimination and aggressiveness via cuticular hydrocarbons in a supercolony-forming ant, Formica yessensis.

BACKGROUND: Territorial boundaries between conspecific social insect colonies are maintained through nestmate recognition systems. However, in supercolony-forming ants, which have developed an extraordinary social organization style known as unicoloniality, a single supercolony extends across large geographic distance. The underlying mechanism is considered to involve less frequent occurrence of intraspecific aggressive behaviors, while maintaining interspecific competition. Thus, we examined whether the supercolony-forming species, Formica yessensis has a nestmate recognition system similar to that of the multicolonial species, Camponotus japonicus with respect to the cuticular hydrocarbon-sensitive sensillum (CHC sensillum), which responds only to non-nestmate CHCs. We further investigated whether the sensory system reflects on the apparent reduced aggression between non-nestmates typical to unicolonial species. METHODOLOGY/PRINCIPAL FINDINGS: F. yessensis constructs supercolonies comprising numerous nests and constitutes the largest supercolonies in Japan. We compared the within-colony or between-colonies' (1) similarity in CHC profiles, the nestmate recognition cues, (2) levels of the CHC sensillar response, (3) levels of aggression between workers, as correlated with geographic distances between nests, and (4) their genetic relatedness. Workers from nests within the supercolony revealed a greater similarity of CHC profiles compared to workers from colonies outside it. Total response of the active CHC sensilla stimulated with conspecific alien CHCs did not increase as much as in case of C. japonicus, suggesting that discrimination of conspecific workers at the peripheral system is limited. It was particularly limited among workers within a supercolony, but was fully expressed for allospecific workers. CONCLUSIONS/SIGNIFICANCE: We demonstrate that chemical discrimination between nestmates and non-nestmates in F. yessensis was not clear cut, probably because this species has only subtle intraspecific differences in the CHC pattern that typify within a supercolony. Such an incomplete chemical discrimination via the CHC sensilla is thus an important factor contributing to decreased occurrence of intraspecific aggressive behavior especially within a supercolony.

Pheromone communication and the mushroom body of the ant, Camponotus obscuripes (Hymenoptera: Formicidae).

Communication by means of pheromones plays predominant roles in colony integration by social insects. However, almost nothing is known about pheromone processing in the brains of social insects. In this study, we successfully applied intracellular recording and staining techniques to anatomically and physiologically characterize brain neurons of the ant Camponotus obscuripes. We identified 42 protocerebral neurons that responded to undecane and/or formic acid, components of alarm pheromones that evoke attraction or evasive behavior, respectively. Notably, 30 (71%) of these neurons were efferent (output) or feedback neurons of the mushroom body, and many of these exhibited different responses to formic acid and undecane. Eight of the remaining 12 neurons had arborizations in the lateral and/or medial protocerebrum, which receive terminations of efferent neurons of the mushroom body and from which premotor descending neurons originate. The remaining four neurons were bilateral neurons that connect lateral accessory lobes or dorsal protocerebrums of both hemispheres. We suggest that the mushroom body of the ant participates in the processing of alarm pheromones. Seventeen (40%) of 42 neurons exhibited responses to nonpheromonal odors, indicating that the pheromonal and nonpheromonal signals are not fully segregated when they are processed in the protocerebrum. This may be related to modulatory functions of alarm pheromones, i.e., they change alertness of the ant and change responses to a variety of sensory stimuli.