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.

Identification of a Male-Produced Aggregation Sex Pheromone in Rosalia batesi, an Endemic Japanese Longhorn Beetle.

The longhorned beetle Rosalia batesi Harold (Coleoptera; Cerambycidae) is endemic to Japan, where its range extends from Hokkaido to Kyushu. The colorful adults are well-known to entomologists and collectors worldwide. It is a hardwood-boring species with larvae that develop in dead broad-leaf trees. In laboratory bioassays, females were attracted to males, which suggested that males produce a sex pheromone. The congeneric species R. alpina is native to Europe, and another congener, R. funebris, is distributed in North America. The pheromone components produced by males of these species had been previously identified as two compounds from different biosynthetic pathways. In the present study, volatiles were collected from beetles of both sexes, and the analyses of the resulting extracts revealed a single male-specific compound, which was identified as 3,5-dimethyl-6-(1-methylbutyl)-pyran-2-one; this is the same compound as the pheromone of the European R. alpina. This alkylated pyrone structure is, so far, unique among known cerambycid pheromones. In field bioassays with traps baited with the racemic synthetic pheromone, significant numbers of both sexes of R. batesi were attracted in an approximately equal ratio, indicating that the compound is an aggregation-sex pheromone rather than a sex pheromone.

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.

Sex Pheromone of the Azalea Mealybug With a Non-Terpene Structure.

Mealybug females release sex pheromones to attract conspecific males for mating. It is critical for mealybug males, which are fragile and short-lived, to respond to the pheromone of their species without time- and energy-consuming cross-attractions to other species. Thus, mealybug pheromone systems are considered to have evolved to be species-specific with unique structures in each species and offer an opportunity to study the diversity of pheromone chemistry that mediates intersexual courtship signals. More than 20 mealybug pheromones are reported to be monoterpenes in general, with only one exception, a hemiterpene alcohol esterified with a medium-chain fatty acid (MCFA), found in the Matsumoto mealybug, Crisicoccus matsumotoi. However, it is unknown whether this is truly exceptional, or if similar compounds are used in other related mealybugs. In this study, we isolated and characterized the pheromone of an allied species, the azalea mealybug C. azaleae. Using gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and bioassays with synthetics, the pheromone was shown to be composed of isopropyl (E)-7-methyl-4-nonenoate, isopropyl (E)-7-methyl-4-octenoate, and ethyl (E)-7-methyl-4-nonenoate. Surprisingly, the structures of these compounds do not include hemiterpene nor monoterpene motifs but have methyl-branched MCFA parts that are similar to an acid moiety of the C. matsumotoi pheromone. This study implies irregular events for the divergence of pheromone structures in ancestors of the genus Crisicoccus and other mealybugs.

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 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.

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.

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.

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.

GABA, ß-alanine and glycine in the digestive juice of privet-specialist insects: convergent adaptive traits against plant iridoids.

The privet tree, Ligustrum obtusifolium (Oleaceae), defends its leaves against insects with a strong lysine-decreasing activity that make proteins non-nutritive. This is caused by oleuropein, an iridoid glycoside. We previously found that some privet-specialist caterpillars adapt by secreting glycine in the digestive juice as a neutralizer that prevents the loss of lysine. Here, we extended the survey into 42 lepidopteran and hymenopteran species. The average concentration of glycine in digestive juice for 11 privet-feeding species (40.396 mM) was higher than that for 32 non-privet-feeding species (2.198 mM). The glycine concentrations exceeded 10 mM in 7 out of 11 privet-feeding species. In Macrophya timida (Hymenoptera), it reached 164.8 mM. Three out of the four remaining privet-feeding species had other amino acids instead. Larvae of a privet-specialist butterfly, Artopoetes pryeri (Lycaenidae), had a high concentration (60.812 mM) of GABA. In two other specialists, ß-alanine was found. GABA, ß-alanine, and glycine as well as alanine, amines, and ammonium ion inhibited the lysine decrease, indicating that amino residues are responsible for the inhibition. However, the three amino acids found in the specialists were far more effective (20 mM showed 80% inhibition) than the rest (>140 mM was required for 80% inhibition). Our results show a clear and rare case of the apparent convergent evolution of herbivores' molecular adaptations of feeding on a plant with a chemical defense in a manner that minimizes the cost of adaptation. The novel role of GABA in plant-herbivore interactions shown here is probably the first reported non-neuronal role of animal-derived GABA.

Sex pheromone and related compounds in the Ishigaki and Okinawa strains of the tussock moth Orgyia postica (Walker) (Lepidoptera: Lymantriidae).

Two distinct electroantennographycally active (EAG-active) components, A and B, and a weakly active component C were found in a solvent extract from virgin females of the Ishigaki strain of the tussock moth, Orgyia postica (Walker). Components A, B, and C were found in the extract of the females at 4.0, 0.5, and 4.0 ng/female respectively. Components A, B, and C were identified as (6Z,9Z,11S,12S)-11,12-epoxyhenicosa-6,9-diene [(11S,12S)-1: posticlure], (6Z)-henicos-6-en-11-one (2), and (6Z,9Z)-henicosa-6,9-diene (3), respectively. Component B was absent in the extract from the Okinawa strain, in which components A and C were present at 2.0 and 1.5 ng/female respectively. (11S,12S)-1 and the racemic mixture showed attractiveness for both the Okinawa and Ishigaki strains, whereas (11R,12R)-1 did not. The addition of 2 significantly reduced the trap catches with (11S,12S)-1 on the Okinawa strain which lacked 2, while there was no significant inhibitory effect on the Ishigaki strain. The addition of 3 to (11S,12S)-1 did not significantly affect trap catches at Ishigaki or Okinawa. This confirmed that the attractant pheromone of O. postica of the Ishigaki strain is also (11S,12S)-1.

Identification of a sex pheromone component of the geometrid moth Milionia basalis pryeri.

A single component in extracts of virgin female Milionia basalis pryeri moths elicited responses from male moth antennae. This compound (ca. 7 ng/female) was identified as (Z,Z)-(3S,4R)-3,4-epoxynonadeca-6,9-diene by GC-MS and NMR analyses, microchemical reactions, and comparative chiral HPLC. In a field test, synthetic (Z,Z)-(3S,4R)-3,4-epoxynonadeca-6,9-diene attracted male moths. The opposite enantiomer, the racemic mixture, and virgin female moths held in small cages attracted no more moths than the solvent controls.

Entomogenous fungus Nomuraea rileyi inhibits host insect molting by C22-oxidizing inactivation of hemolymph ecdysteroids.

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae.