Jun Tabata

Functional consequences of sequence variation in the pheromone biosynthetic gene pgFAR for Ostrinia moths

Pheromones are central to the mating systems of a wide range of organisms, and reproductive isolation between closely related species is often achieved by subtle differences in pheromone composition. In insects and moths in particular, the use of structurally similar components in different blend ratios is usually sufficient to impede gene flow between taxa. To date, the genetic changes associated with variation and divergence in pheromone signals remain largely unknown. Using the emerging model system Ostrinia, we show the functional consequences of mutations in the protein-coding region of the pheromone biosynthetic fatty-acyl reductase gene pgFAR. Heterologous expression confirmed that pgFAR orthologs encode enzymes exhibiting different substrate specificities that are the direct consequences of extensive nonsynonymous substitutions. When taking natural ratios of pheromone precursors into account, our data reveal that pgFAR substrate preference provides a good explanation of how species-specific ratios of pheromone components are obtained among Ostrinia species. Moreover, our data indicate that positive selection may have promoted the observed accumulation of nonsynonymous amino acid substitutions. Site-directed mutagenesis experiments substantiate the idea that amino acid polymorphisms underlie subtle or drastic changes in pgFAR substrate preference. Altogether, this study identifies the reduction step as a potential source of variation in pheromone signals in the moth genus Ostrinia and suggests that selection acting on particular mutations provides a mechanism allowing pheromone reductases to evolve new functional properties that may contribute to variation in the composition of pheromone signals.

Male killing and incomplete inheritance of a novel spiroplasma in the moth Ostrinia zaguliaevi.

Bacteria of the genus Spiroplasma are widely found in plants and arthropods. Some of the maternally transmitted Spiroplasma endosymbionts in arthropods are known to kill young male hosts (male killing). Here, we describe a new case of Spiroplasma-induced male killing in a moth, Ostrinia zaguliaevi. The all-female trait caused by Spiroplasma was maternally inherited for more than 11 generations but was spontaneously lost in several lineages. Antibiotic treatment eliminated the Spiroplasma infection and restored the 1:1 sex ratio. The survival rates and presence/absence of the W chromosome in the embryonic and larval stages of O. zaguliaevi showed that males were selectively killed, exclusively during late embryogenesis in all-female broods. Based on phylogenetic analyses of 16S rRNA, dnaA and rpoB gene sequences, the causative bacteria were identified as Spiroplasma belonging to the tick symbiont Spiroplasma ixodetis clade. Electron microscopy confirmed bacterial structures in the follicle cells and follicular sheath of adult females. Although many congeneric Ostrinia moths harbor another sex ratio-distorting bacterium (Wolbachia), only O. zaguliaevi harbors Spiroplasma.

Variation in Courtship Ultrasounds of Three Ostrinia Moths with Different Sex Pheromones

Moths use ultrasounds as well as pheromones for sexual communication. In closely related moth species, variations in ultrasounds and pheromones are likely to profoundly affect mate recognition, reproductive isolation, and speciation. The European corn borer, Ostrinia nubilalis, and its Asian congeners, Ostrinia furnacalis and Ostrinia scapulalis, exhibit within-species and between-species variation in their pheromone communication. Recently, we reported ultrasound communication in O. furnacalis; however, variations in ultrasounds in the three congeners have not been addressed to date. Here we investigated features of ultrasound production and hearing in O. nubilalis and O. scapulalis, and compared them with those of O. furnacalis. As in O. furnacalis, males of O. nubilalis and O. scapulalis produced ultrasounds during courtship by rubbing specialized scales on the wings against scales on the thorax. The covering of these scales with nail polish muffled the sounds and significantly reduced mating success in O. nubilalis, showing the importance of ultrasound signaling in mating. The ultrasounds produced by O. nubilalis and O. scapulalis were similar, consisting of long trains of pairs of pulses with a main energy at 40 kHz, but distinctly different from the ultrasound produced by O. furnacalis, consisting of groups of pulses peaking at 50 kHz and with substantially more energy up to 80 kHz. Despite overall similarities, temporal features and patterns of amplitude modulation differed significantly among the geographic populations of O. nubilalis and O. scapulalis, which differed in pheromone type. In contrast, no significant difference in hearing was found among the three species with regard to the most sensitive frequencies and hearing threshold levels. The patterns of variations in the songs and pheromones well reflected those of the phylogenetic relationships, implying that ultrasound and pheromone communications have diverged concordantly. Our results suggest that concordant evolution in sexual signals such as courtship ultrasounds and sex pheromones occurs in moths.

GENETIC BASIS TO DIVERGENCE OF SEX PHEROMONES IN TWO CLOSELY RELATED MOTHS, Ostrinia scapulalis AND O. zealis

Crossing experiments between two closely related moths, Ostrinia scapulalis and O. zealis, were conducted to gain insight into the genetic basis of the divergence of female sex pheromones. The sex pheromone of O. scapulalis comprises (E)-11- and (Z)-11-tetradecenyl acetates (E11 and Z11), and distinct genetic variation is found in the blend of components. This variation is largely controlled by a single autosomal locus with two alleles, AE(sca) and AZ(sca). E-type (AE(sca)AE(sca)) females produce a pheromone with amean E11:Z11 ratio of 99:1, whereas Z-type (AZ(sca)AZ(sca)) and I-type (AE(sca)AZ(sca)) females produce a pheromone with a mean of 3:97 and 64:36, respectively. O. zealis is distinctive in that it has a third pheromone component, (Z)-9-tetradecenyl acetate (Z9), in addition to E11 and Z11, and the typical blend ratio is 60:35:5 (Z9:E11:Z11). Our study revealed that Z9 production in O. zealis is mainly regulated by an autosomal recessive gene phr(zea), which is suggested to be involved in the chain-shortening of a pheromone precursor fatty acid, and linked to AE(zea), a gene corresponding to AE(sca) in O. scapulalis. A few mutations in a gene involved in pheromone production could explain the dramatic shift between a two-component pheromone communication system in O. scapulalis and a three-component system in O. zealis.

Sex pheromone production and perception in the mating disruption‐resistant strain of the smaller tea leafroller moth, Adoxophyes honmai

Mating disruption is an environmentally safe plant protection strategy that uses a synthetic copy of an insect pheromone to interfere with sexual communication and hence reproduction. To date, a number of pest moths have been controlled with applications of formulated pheromones as mating disruptants. Recently, however, the first example of resistance to mating disruption was documented in one of the major tea pests in Japan, the smaller tea leafroller moth, Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae). To avoid other such cases, it is important to elucidate the mechanism(s) by which the disruptant lost its effectiveness. To this end, we imposed further selection by rearing field‐collected resistant insects with a synthetic pheromone in the laboratory. After more than 70 generations of selection, a strain with quite strong resistance was established, males of which could find and copulate with their mates even in the presence of 1 mg l−1 of disruptant. Although the mating ability of this strain was greatly increased, the composition and blend ratio of the sex pheromone produced and emitted by females were not obviously changed in comparison with those of females sensitive to mating disruption. However, male response to the pheromone blend was markedly broadened after selection so that resistant males could locate a synthetic pheromone source even when it lacked a pheromone component that is normally necessary for attraction. Males capable of locking onto off‐ratio pheromone blends may be better able to find calling females in pheromone‐treated environments than narrowly tuned males because of greater capability of overcoming sensory imbalance.

Cyclolavandulyl butyrate: an attractant for a mealybug parasitoid, Anagyrussawadai (Hymenoptera: Encyrtidae)

In this study, we discovered and isolated an attractant for a mealybug-parasitic wasp Anagyrussawadai from an esterification product prepared with commercialized lavandulol (2-isopropenyl-5-methyl-4-hexen-1-ol) and butyryl chloride. Using gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy, we determined the structure of the compound to be (2,4,4-trimethyl-2-cyclohexenyl)methyl butyrate (cyclolavandulyl butyrate). This is a novel compound as far as we know, although the alcohol moiety has been known as a cyclization product from lavandulol. Cyclolavandulyl butyrate has two enantiomers, and the (−)-isomer, which is suggested to have S configuration, showed higher attractiveness. A potential use for the A. sawadai attractant for mealybug management in agricultural fields is discussed.

Female sex pheromone of Ostrinia orientalis – throwing a light on the relationship between O. orientalis and the European corn borer, O. nubilalis

Summary.The sex pheromone of Ostrinia orientalis (Lepidoptera: Crambidae) was analyzed by gas chromatography–electroantennographic detection (GC–EAD), GC–mass spectrometry and a series of bioassays. Three EAD-active compounds were detected in the female sex pheromone gland extract, and identified as tetradecyl acetate (14:OAc), (Z)-11-tetradecenyl acetate (Z11-14:OAc) and (E)-11-tetradecenyl acetate (E11-14:OAc). The titers (ratio) of 14:OAc, Z11-14:OAc and E11-14:OAc in 3-day-old virgin females were 0.49 ng (10), 4.86 ng (98) and 0.10 ng (2), respectively. In a wind-tunnel bioassay, the 98:2 blend of Z11- and E11-14:OAc, but not Z11-14:OAc alone, elicited the same male behavioral responses as virgin females and crude gland extracts. 14:OAc was inactive by itself, and did not show any synergistic effect on the binary blend. Field trapping experiments also confirmed the attractiveness of the binary blend to O. orientalis males. Based on these results, we concluded that the sex pheromone of O. orientalis is a 98:2 mixture of Z11-14:OAc and E11-14:OAc. This sex pheromone is very similar to that of the Z-type European corn borer, O. nubilalis. The present finding raises the question of whether O. orientalis , which is indistinguishable from O. nubilalis based on external morphology, is a biologically distinct species independent from O. nubilalis.

Pheromone analysis of wild female moths with a PBAN c-terminal peptide injection for an estimation of assortative mating in Adzuki bean borer, Ostrinia scapulalis

The adzuki bean borer, Ostrinia scapulalis, has distinct genetic variation in the blend of two sex pheromone components, (E)- and (Z)-11-tetradecenyl acetates. This variation is largely controlled by a single autosomal locus with two alleles, E and Z. E-type (EE) females produce a pheromone with a mean E:Z ratio at 99:1 whereas Z-type (ZZ) and I-type (ZE) produce pheromones with mean of 3:97 and 64:36, respectively. Interestingly, in many natural populations of O. scapulalis in Japan, this pheromone polymorphism appears to be stably maintained. We tried to predict the changes in relative abundance of each pheromone type by estimating the pheromone production genotype of wild females and their male mates. The pheromone titer in the wild, mated females was increased without changing the blend ratio by an injection of a peptide with pheromone biosynthesis activating activity (TKYFSPRL-NH2). The frequencies of E-, I-, and Z-types at Matsudo were 15, 52, and 33%, respectively, and did not deviate from the Hardy–Weinberg expectations. The estimated mating patterns were concordant with the assumption that no assortative mating was occurring in this population, and this is suggested as a cause of sustained polymorphism at Matsudo.

A new approach for mealybug management: recruiting an indigenous, but ‘non‐natural’ enemy for biological control using an attractant

We previously discovered that (2,4,4‐trimethyl‐2‐cyclohexenyl)‐methyl butyrate (cyclolavandulyl butyrate, CLB) is an attractant for the mealybug‐parasitic wasp Anagyrus sawadai Ishii (Hymenoptera: Encyrtidae: Anagyrini). This wasp is not likely to parasitize the Japanese mealybug, Planococcus kraunhiae (Kuwana) (Hemiptera: Pseudococcidae), under natural conditions. In this study, we showed that this ‘non‐natural’ enemy wasp can parasitize P. kraunhiae in the presence of CLB in field experiments. Laboratory‐reared mealybugs placed on persimmon trees with CLB‐impregnated rubber septa were parasitized significantly more often by endoparasitic wasps than those on non‐treatment trees (18.1–40.3 vs. 0–6.3%). Anagyrus sawadai accounted for 20% of the wasps that emerged from mealybugs placed on CLB‐treated trees. Moreover, CLB attracted another minor parasitoid, Leptomastix dactylopii Howard (Hymenoptera: Encyrtidae: Anagyrini), which also parasitized more P. kraunhiae in the presence of CLB. All wasps that emerged from the mealybugs on control trees were Anagyrus fujikona Tachikawa, a major parasitoid of P. kraunhiae around the test location. These results demonstrated that CLB can recruit an indigenous, but ‘non‐natural’ enemy that does not typically attack P. kraunhiae under natural conditions, as well as a minor natural enemy, for biological control of this mealybug species.

Oviposition-Stimulatory Activity against Ostrinia Zealis by Essential Oil of Root Part from Cirsium Japonicum DC

The essential oil components from root part of Cirsium japonicum and oviposition-stimulatory activity against Ostrinia zealis by root essential oil were investigated. The main component of fresh root essential oil was aplotaxene (75.14%) and of dried root stock was found to be palmitic acid (14.42%). The root oil was significantly more active than the control against O. zealis.