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The smaller tea tortrix, Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae), is one of the main insect pests of tea, Camellia sinensis Kuntz, in Japan. Recently, A. honmai has developed a high resistance to diacylhydrazine analog insect growth regulators, such as tebufenozide, in Shizuoka Prefecture. Previously, we identified a point mutation (A415V) in the ecdysone receptor gene (EcR), a candidate factor responsible for tebufenozide resistance. In this study, we have developed a molecular method of diagnosis to detect the EcR A415V mutation by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). This method was confirmed to be successfully applicable to larvae reared in the laboratory and adults collected by pheromone traps in the field. The appearance ratio of the resistant allele in the A. honmai populations from various Japanese districts examined by the method revealed a high correlation with the magnitude of tebufenozide resistance. These results verified that the A415V mutation is the principal factor responsible for tebufenozide resistance and the PCR–RFLP method may be used as a reliable and convenient tool for monitoring tebufenozide resistance in the field.

The smaller tea tortrix, Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae), is one of the most common pests that affect the tea plant, Camellia sinensis (L.) O. Kuntze. Diacylhydrazine-analog insect growth regulators, such as tebufenozide, have been used as controls; however, the species began showing resistance to this compound in approximately 2004. Previously, we identified an amino acid mutation (A415V) in the ecdysone receptor gene (EcR) as a principal factor in tebufenozide resistance and developed a molecular diagnostic method to detect the EcR A415V mutation through polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). However, for the continuous management of tebufenozide resistance in A. honmai, a simple and quick genetic diagnostic method that does not require specialized equipment is preferable. In this study, we developed a novel technique to detect the mutation using allele-specific loop-mediated isothermal amplification (AS-LAMP). This method enables the monitoring and detection of tebufenozide-resistance alleles much faster than PCR–RFLP and with moderate accuracy. Therefore, it could be a powerful tool for the early detection of tebufenozide-resistant A. honmai in tea plantations.

Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae) is a serious pest of tea plants in Japan. Damage caused by larvae is widely distributed throughout tea plantations, but the modes of oviposition and offspring feeding are still unclear. Therefore, the objective of the present study was to determine oviposition preference for leaf age in A. honmai as related to performance of offspring. Two-choice experiments between young and old leaves showed that A. honmai female adults preferred to oviposit on the area close to old leaves rather than young leaves. Therefore, volatiles from old tea leaves appear to act as attractants and stimulate oviposition. In contrast, more than 70 % of neonates fed on young leaves 24 h after starting the experiments, and development of larvae that continuously fed on young leaves was higher than larvae that fed on old leaves. After hatching, neonates likely move to the young leaves to start feeding. Finally, we conclude that leaf age affects not only maternal insect ability to find suitable habitats for oviposition, but also affects the development of their offspring.

We investigated the susceptibility of the smaller tea tortrix, Adoxophyes honmai Yasuda, to diamide insecticides in the Shimada-Yui tea fields in Shizuoka Prefecture, Japan, from 2006 to 2011. By 2011, the insects had developed significant resistance even to concentrations far above the registration concentrations of two diamides, flubendiamide and chlorantraniliprole. The lethal concentration 50 (LC50) values of flubendiamide showed a rapid annual increase from 16.2 ppm in 2007 to 161 ppm in August 2011, exceeding the registration concentration of 100 ppm in 2010 and 2011. The LC50 values of chlorantraniliprole increased sharply from 25.3 ppm in 2010 to 98.8 ppm in August 2011, exceeding the registration concentration of 50 ppm. The LC50 values for flubendiamide and chlorantraniliprole at 10 days after treatment in insects collected in August 2011 were 105-fold and 77.2-fold higher, respectively, than those in a susceptible strain.

In Adoxophyes honmai, a population that has developed resistance to a disruptant composed of (Z)-11-tetradecenyl acetate was recognized in Shimada in Shizuoka Prefecture. The sex pheromone components of the resistant population were individually analyzed. The average amounts of (Z)-9- and (Z)-11-tetradecenyl acetates were 40.5 ng and 45.6 ng, respectively, whereas the averages in the susceptible population were 39.9 ng and 15.1 ng. The results suggest that resistant females biosynthesize more (Z)-11-tetradecenyl acetate in order to heighten the ratio of the acetate in the sex pheromone component. A 40 : 60 blend of (Z)-9- and (Z)-11-tetradecenyl acetates was the best of six kinds of lures tested on resistant males. The male response tunes to the sex pheromone blend produced by resistant females. Although no attractiveness has been generally observed in a lure impregnated with only (Z)-9-tetradecenyl acetate, males were attracted to the incomplete lure treated with the mating disruptant in the field. Resistant males supposedly utilize the widespread odor of (Z)-11-tetradecenyl acetate released from mating disruption devices installed in the tea canopy for orientation, while they trace a directional plume of (Z)-9-tetradecenyl acetate emitted from the lure.

The effects of associative learning of plant chemicals on host-searching behavior in Ascogaster reticulata Watanabe, an egg-larval parasitoid of the smaller tea tortrix, Adoxophyes honmai Yasuda, were investigated. Learned responses of conditioned and non-conditioned plant species were examined by a two-choice assay. Before the two-choice assays, females were given a rewarding oviposition experience or unrewarding experience on a leaf disc or leaf extract of either tea, camellia, sasanqua, bayberry, chinquapin, rose, Japanese cedar, fern pine, mulberry and corn. Following the experience, females were tested in a choice test between tea leaf and an alternative plant leaf. The results of the two-choice assays with leaf square discs were similar to tests using leaf extracts. Females that were conditioned with tea leaf showed a significant preference for tea leaf over the alternative plant species, except for leaves of the genus Camellia (camellia and sasanqua). Wasps conditioned with the other nine plant leaves only showed a significant preference for bayberry, mulberry, fern pine and corn. Females that had rewarding and unrewarding experiences were able to distinguish between tea and camellia. These results suggest that A. reticulata females are able to distinguish different plant species except for closely related species and some plant species; however, combinations with rewarding and unrewarding experiences enabled females to discriminate closely related species.

The late male-killing phenomenon was reported in the oriental tea tortrix, Homona magnanima, but its gross pathology and histopathology have not been elucidated. The present study investigated pathological changes in larvae of a late male-killing strain of H. magnanima (LMK). Most male LMK larvae died during the last instar or pupal stage, and dead male larvae showed characteristic signs and symptoms of male killing. The body color of male LMK larvae started to change 4 d after molting into the fifth instar and became increasingly opaque white. Male LMK larvae weighed significantly less than normal-strain (NSR) males. Midgut epithelial cells of male LMK larvae developed normally and microvilli were observed on the luminal side 3 d after the final molt. Regenerative cells of male LMK larvae were observed on the basement membrane but these cells did not develop to pupal midgut cells. From 7 d after the final molt, midgut cells of male LMK larvae were discharged into the lumen as granules and regenerative cells elongated from the basement membrane. All midgut cells of male LMK larvae dropped into the lumen just before death and no cells were observed on the basement membrane. The fat body of male LMK larvae contained large fat granules in the cytoplasm but became a tumor-like cell mass that finally fused with epidermal cells. These changes were specific to LMK males; no differences in the developmental pattern or morphology were observed between female LMK and NSR larvae. From these results, it is suggested that the degradation of midgut epithelial cells may be the main reason for late male killing.

The first example of resistance to a mating disruptant was reported in the smaller tea tortrix moth, Adoxophyes honmai. The active ingredient in the disruptant was (Z)-11-tetradecenyl acetate (Z11-14:OAc), which is one component of the sex pheromone of A. honmai. From a resistant colony in the field, a strain highly resistant to mating disruption was established through further selection imposed in the laboratory by rearing in the presence of Z11-14:OAc. With this resistant (R) strain, we investigated the response of males to mixtures of synthetic pheromone components in the present study. Males of the R strain were less attracted to a low concentration of the pheromone than non-resistant (S) males. However, the R males were attracted to lures with extremely deviated ratios of the pheromone components: 72% of them even responded to the mixture lacking Z11-14:OAc, which is indispensable for response behavior of the S males. Some F1 males of crosses between S and R (13–15%) and male progeny in both reciprocal backcrosses to R (29–38%) were also attracted to the mixture without Z11-14:OAc. The results of these crossing experiments could be seen corresponding with the expected pattern in a single-autosomal-locus model for inheritance of the ability to respond to a defective pheromone blend.

At tea fields in Shimada in Shizuoka Prefecture, a mating disruptant composed of (Z)-11-tetradecenyl acetate (trade name: Hamaki-con) had been used for controlling Adoxophyes honmai (Yasuda) annually since 1983. In 1986, four years after use of the disruptant was initiated, the disruption percentage of pheromone trap catches was 96%, and a high control effect was observed. However, from 1996 to 1998, 14 to 16 years after treatment was started, the percentages became less than 50%. When the disruptant was applied in other tea fields where it had not been previously used, the disruption percentages were over 99%. These results strongly suggest the possibility that a selection pressure caused by continuous use of the disruptant should have caused the low disruption percentage in Shimada. We propose that the phenomenon be termed “resistance” to a mating disruptant composed of (Z)-11-tetradecenyl acetate in A. honmai. When the sex pheromone blend of A. honmai, 63 : 31 : 4 : 2 mixture of (Z)-9-tetradecenyl acetate, (Z)-11-tetradecenyl acetate, (E)-11-tetradecenyl acetate and 10-methyldodecyl acetate (4-component blend), was applied as a disruptant for the “resistance” population, the disruption percentage was 99% and the larval density in the next generation decreased. These results suggest that the 4-component blend is useful as a control tool for the “resistance” population.

The mariner-like element (MLE) in two closely related leaf-roller moths, the smaller tea tortrix, Adoxophyes honmai and the summer fruit tortrix, Adoxophyes orana fasciata (Lepidoptera: Tortricidae) were surveyed. By a series of PCR and Southern blotting experiments, there were at least two types of MLEs in these two species. Sequence dissimilarity of each MLE suggests a possible polyphyletic origin and subsequent vertical inheritance within each species. In addition, a full-length MLE was isolated from a genomic library of A. honmai. This element, named Ahmarl, was 1,294 bp long with 27 bp inverted terminal repeats (ITRs). This element is apparently inactive due to a frameshift in the open reading frame (ORF) region, but a putative ORF could be observed by a single nucleotide removal, suggesting that the MLE in this species could have had the ability to transpose in the near past. Ahmarl may be suitable as a transformation vector with slight modification.