Evolutionary origin and distribution of amino acid mutations associated with resistance to sodium channel modulators in onion thrips, Thrips tabaci.

In onion thrips Thrips tabaci, reduced sensitivity of the sodium channel caused by several sodium channel mutations have been correlated with pyrethroid resistance. For this study, using mitochondrial cytochrome c oxidase subunit I gene sequences, we examined the phylogenetic relation among a total of 52 thelytokous and arrhenotokous strains with different genotypes of the sodium channel mutations. Then, we used flow cytometry to estimate their ploidy. Results showed that the strains are divisible into three groups: diploid thelytoky, triploid thelytoky, and diploid arrhenotoky. Using 23 whole genome resequencing data obtained from 20 strains out of 52, we examined their genetic relation further using principal component analysis, admixture analysis, and a fixation index. Results showed that diploid and triploid thelytokous groups are further classifiable into two based on the sodium channel mutations harbored by the respective group members (strains). The greatest genetic divergence was observed between thelytokous and arrhenotokous groups with a pair of T929I and K1774N. Nevertheless, they shared a genomic region with virtually no polymorphism around the sodium channel gene loci, suggesting a hard selective sweep. Based on these findings, we discuss the evolutionary origin and distribution of the sodium channel mutations in T. tabaci.

Diversity and composition of flower-visiting insects and related factors in three fruit tree species.

Animal-mediated pollination is an essential ecosystem service for the production of many fruit trees. To reveal the community composition of flower-visiting wild insects which potentially contribute to fruit production and to examine the effects of geographic location, local meteorological conditions and locally introduced domesticated pollinators on them, we investigated the community composition of insects visiting the flowers (hereafter, "visitors") of apple, Japanese pear and Oriental persimmon for 1‒3 years at 20 sites around Japan. While most of the variation (82%) of the community composition was explained by tree species with a slight contribution by geographic distance (2%), maximum temperature and tree species contributed 62% and 41% of the variation in total abundance of the visitors, respectively. Though the dominant families of the visitors varied spatiotemporally, the community composition of the visitors of apple and Japanese pear clearly differed from that of Oriental persimmon. While Andrenidae and Syrphidae together accounted for 46%‒64% of the visitors of apple and Japanese pear, Apidae represented 57% of the visitors of Oriental persimmon. The taxonomic richness, diversity and evenness of the visitors were best predicted by locally introduced domesticated pollinators and local meteorological conditions of wind speed and maximum temperature. Amongst these selected factors, locally introduced domesticated pollinators could have the largest impact. It seemed to be strongly related to the reduction of taxonomic richness, diversity and evenness of the visitors, accounting for 41‒89% of the variation. Results suggested that the community composition and total abundance of potential pollinators were predominantly determined by tree species and temperature, but locally introduced domesticated pollinators could have a determinantal pressure on the taxonomic diversity of the community.

Sodium channel mutations (T929I and F1534S) found in pyrethroid-resistant strains of the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae).

RNA-seq data analysis of cigarette beetle (Lasioderma serricorne) strains having different sensitivities to pyrethroids identified sodium channel mutations in strains showing pyrethroid resistance: the T929I and F1534S mutations. These results suggest that reduced sensitivity of the sodium channel confers the pyrethroid resistance of L. serricorne. Results also showed that the F1534S mutation mostly occurred concurrently with the T929I mutation. The functional relation between both mutations for pyrethroid resistance is discussed.

Host plant and humidity effects on phytoseiid mite, Gynaeseius liturivorus (Acari: Phytoseiidae) egg hatchability.

Gynaeseius liturivorus (Ehara) (Acari: Phytoseiidae) is a promising biological control agent for tiny arthropod pests, including Thrips tabaci Lindeman (Thysanoptera: Thripidae) known as a major pest of Welsh onion. In fields during summer, G. liturivorus is observed on soybean, but not on Welsh onion, even when numerous T. tabaci are present. To elucidate G. liturivorus's occurrence on soybean and Welsh onion in relation to relative humidity (RH), we examined its egg hatching on their seedlings under low RH conditions. Then we estimated the moisture transpiration from both plants. Egg hatching occurred only on soybean plants exhibiting greater moisture transpiration. Aiming at utilizing G. liturivorus as a biological control agent for Welsh onion production in summer, evaluation of its relative tolerance and compensation potential for drought injury is necessary. Therefore, we used five phytoseiid species including G. liturivorus and Neoseiulus californicus to estimate the RH and vapor pressure deficit (VPD) at which 50% of eggs hatch (RH50 and VPD50). Furthermore, we examined G. liturivorus and N. californicus egg hatching under different RH oscillation conditions. Results show G. liturivorus as the most drought-sensitive among the five species tested, but G. liturivorus is able to compensate for lethal low-RH effects on egg hatching in part by periodic exposure to high RH conditions, as observed for N. californicus.

Origin, selection, and spread of diamide insecticide resistance allele in field populations of diamondback moth in east and southeast Asia.

BACKGROUND: The investigation of molecular mechanisms and evolution of resistance to insecticides is an ongoing challenge, as researchers must provide guidance to manage the resistance to achieve sustainable production in agriculture. Predicting, monitoring, and managing insecticide resistance requires information on the origins, selection, and spread of resistance genes. The resistance of Plutella xylostella (L.) against diamide insecticides is becoming an increasingly severe problem in east and southeast Asia. In this study, the evolution of resistance was investigated using a resistance allele [ryanodine receptor (RyR); G4946E mutation] and its flanking regions, as well as mitochondrial cytochrome c oxidase subunit I (mtCOI). RESULTS: The sequences of the flanking region of the G4946E and mtCOI suggested that the G4946E mutation has a key role in resistance. Furthermore, the G4946E mutation has multiple origins, and congenic resistant mutations have spread across east and southeast Asia, despite substantial geographical barriers. In addition, the susceptibility of field populations partially recovered during winter, based on the observed decrease in the G4946E (resistant allele) frequency. Finally, the resistance level indexed by the frequency of the E4946 allele was significantly lower in non-overwintering regions than in overwintering regions. CONCLUSION: The information of the present study is useful to monitor resistance using molecular markers and to develop strategies to delay the evolution of diamide resistance.

Estimating the proportion of resistance alleles from bulk Sanger sequencing, circumventing the variability of individual DNA.

Specimens should be examined as much as possible to obtain a precise estimate of the proportion of resistance alleles in agricultural fields. Monitoring traps that use semiochemicals on sticky sheets are helpful in this regard. However, insects captured by such traps are ordinarily left in the field until collection. Owing to DNA degradation, the amount of DNA greatly varies among insects, causing serious problems in obtaining maximum likelihood estimates and confidence intervals of the proportion of the resistance alleles. We propose a statistical procedure that can circumvent this degradation issue. R scripts for the calculation are provided for readers. We also propose the utilization of a Sanger sequencer. We demonstrate these procedures using field samples of diamide-resistant strains of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). The validity of the assumptions used in the statistical analysis is examined using the same data.

Detection of Fusarium oxysporum f. sp. fragariae by Using Loop-Mediated Isothermal Amplification.

We developed a loop-mediated isothermal amplification (LAMP) assay for detecting Fusarium oxysporum f. sp. fragariae, the causal agent of wilt in strawberry plants. This assay was based on genomic regions between the portions of transposable elements Han and Skippy of the fungus. The LAMP assay allowed the efficient detection of F. oxysporum f. sp. fragariae DNA by visual inspection, without requiring gel electrophoresis. The detection limit was 100 pg of genomic DNA, which is comparable to that of PCR. The LAMP primers successfully discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and other fungi. The LAMP assay at 63°C, which was found to be the optimal treatment temperature, for 1.5 h successfully detected F. oxysporum f. sp. fragariae California strains GL1270 and GL1385. When the assay was performed using a Genelyzer FIII portable fluorometer, these California strains were successfully detected in 1 h. The assay facilitated the detection of conidia in soil samples after they were precultured on a selective medium for F. oxysporum (FoG2) as well as latent infection in strawberry plants after preculturing. The LAMP assay for visual inspection of DNA required only a heating block and an incubator, reducing the cost of this assay. Thus, it could be suitable for the detection of F. oxysporum f. sp. fragariae strains in centers that store prefoundation and foundation stocks of strawberry, including plant nurseries.

Field survey of ryanodine receptor mutations (G4946E and I4790K) and their effects on biotic performance in the diamondback moth.

This study examined changes in the proportions of ryanodine receptor mutations (G4946E and I4790K) for Plutella xylostella populations in the field. Results show that the proportion of G4946E decreased during the survey years without diamide application and that insects with I4790K were observed less frequently during the survey period. This study also examined the biotic performances of diamide-resistant P. xylostella strains. The results show that the diamide-resistant strains exhibited hatchability, larval development, and fecundity equivalent to those of diamide-susceptible strains.

Molecular monitoring of Neoseiulus californicus released from sheltered slow-release sachets for spider mite control in a Japanese pear greenhouse.

A novel system for spider mite control was developed with a slow-release sachet containing Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) protected by a waterproof shelter. Monitoring the efficacy of the predator release system for spider mite control at a Japanese pear greenhouse requires discrimination of N. californicus from other indigenous phytoseiid mite species inhabiting the study site and subsequent identification of the released N. californicus. The report of our earlier study described a PCR-based method for discrimination of N. californicus species. For the present study, we first examined phytoseiid mite species composition in the greenhouse. Subsequently, we developed microsatellite markers to identify the released N. californicus. Finally, we installed the predator release system in the greenhouse and conducted a population survey of phytoseiid and spider mites. Results demonstrated that approximately 1 month is necessary for distribution of the released N. californicus on the leaves.

Long-term field insecticide susceptibility data and laboratory experiments reveal evidence for cross resistance to other neonicotinoids in the imidacloprid-resistant brown planthopper Nilaparvata lugens.

BACKGROUND: Long-term monitoring data is helpful to understand the fluctuation of susceptibility and pattern of cross resistance in insecticide resistance management. After the occurrence of imidacloprid resistance, the brown planthopper (BPH) has gradually developed resistance to thiamethoxam and clothianidin since 2010, but not to dinotefuran and nitenpyram. Here, we analyzed susceptibilities data of five neonicotinoids during 2005-2017 in East Asia and Vietnam to conduct cross-resistance patterns among neonicotinoids. To determine the factors of development of cross resistance in laboratory bioassays, we used the imidacloprid resistant and control strains that were selected from filed populations in the Philippines and Vietnam. RESULTS: The Linear Mixed Models (LMM) analyses of insecticide susceptibility data showed that the slope values of imidacloprid resistance effects were 0.68 and 1.09 for resistance to thiamethoxam and clothianidin, respectively. Laboratory bioassay results showed that the LD50 values for thiamethoxam and clothianidin in resistant strains (1.4-5.5 µg g-1 ) were 3.2-16.4 times higher than those in the control strains (0.28-1.5 µg g-1 ). However, the increase in the LD50 values for imidacloprid was not related to that for dinotefuran and nitenpyram based on the results of the LMM analysis and laboratory bioassay. CONCLUSION: Our results demonstrate that the development of imidacloprid resistance result in strong-cross resistance to some neonicotinoids, thiamethoxam and clothianidin, but not to others, dinotefuran and nitenpyram. We anticipate that our findings will be a starting point for understanding mechanism of the different trend of cross resistance by analyzing long-term susceptibility data and laboratory bioassays in insect pests. © 2019 Society of Chemical Industry.

Ryanodine receptor mutations (G4946E and I4790K) differentially responsible for diamide insecticide resistance in diamondback moth, Plutella xylostella L.

This study examined diamondback moth (Plutella xylostella) strains showing high-level resistance to cyantraniliprole (KA17 strain) and to flubendiamide and chlorantraniliprole (KU13 strain). The LC50 value of the KA17 strain against cyantraniliprole was ca. 100-fold higher than that of the KU13 strain. The KA17 strain also exhibited high-level resistance to chlorantraniliprole and flubendiamide equivalent to those in the KU13 strain. The KU13 strain showed a higher LC50 value against cyantraniliprole than the susceptible strains. However, the LC50 value of the KU13 strain against cyantraniliprole was below the agriculturally recommended concentration. Subsequent QTL analysis using ddRAD-seq identified the resistance responsible regions of the KA17 and KU13 strains with different diamide resistance profiles. Ryanodine receptor (RyR) gene was included in the identified regions. Single nucleotide polymorphism calling in the RyR gene using RNA-seq found previously reported G4946E (amino acid mutation from glycine to glutamic acid at amino acid position 4946) and novel I4790K (amino acid mutation from isoleucine to lysine at amino acid position 4790) mutations, respectively, in the RyR of the KU13 and KA17 strains. Functional significance of I4790K in the resistance was confirmed in calcium imaging of the human embryonic kidney 293T cell line expressing Bombyx mori RyR with the mutation. This reporting is the first describing I4790K as a fundamental mechanism responsible for the resistance to the diamides including cyantraniliprole. From this study, we also report up-regulated expression of some degradation enzymes and that of the RyR gene in the KA17 and KU13 strains based on results of RNA-seq data analysis.

Nicotinic acetylcholine receptor α6 subunit mutation (G275V) found in a spinosad-resistant strain of the flower thrips, Frankliniella intonsa (Thysanoptera: Thripidae).

The flower thrips Frankliniella intonsa strain showing resistance to spinosad was established in the laboratory. The resistant strain showed an LC50 value of 1398.7 mg/L in a leaf dipping/contact assay. The LC50 value was ca. 280 times higher than that of the most susceptible strain. An insecticidal assay using synergists suggested no involvement of degradation enzymes, such as cytochrome P450, glutathione S-transferase, and carboxyl esterase, in the resistance. Glycine at amino acid position 275 of the nicotinic acetylcholine receptor (nAChR) α6 subunit was mutated to valine in the resistant strain. These results suggest that spinosad resistance in F. intonsa is conferred by the reduced sensitivity of nAChR.

Insecticide susceptibilities of the two rice planthoppers Nilaparvata lugens and Sogatella furcifera in East Asia, the Red River Delta, and the Mekong Delta.

BACKGROUND: The two rice planthoppers, Nilaparvata lugens and Sogatella furcifera, have different life cycles in the regions of East Asia, the Red River Delta, and the Mekong Delta. The susceptibilities of these species to a range of insecticides have not previously been compared among the three regions over multiple years. Here, we describe the differences and similarities in insecticide susceptibilities of the two species among the three regions in 2006-2011. RESULTS: In all three regions in 2006 - 2011, N. lugens developed high and moderate levels of resistance to imidacloprid and thiamethoxam, respectively, but this species did not develop resistance to fipronil. In contrast, S. furcifera developed a high level of resistance to fipronil. The ranges in 50% lethal dose (LD50 ) values for N. lugens treated with both imidacloprid and thiamethoxam were similar over time between East Asia and the Red River Delta, and were different in the Mekong Delta. CONCLUSION: The results support the idea that resistant populations migrate from the Red River Delta region to East Asia. Therefore, continuous monitoring of the susceptibility of N. lugens to insecticides in the Red River Delta is very important for insecticide resistance management in East Asia. © 2017 Society of Chemical Industry.

Seasonal proportion change of ryanodine receptor mutation (G4946E) in diamondback moth populations.

This study examined the seasonal proportion change of ryanodine receptor mutation (G4946E) for Plutella xylostella populations using quantitative sequencing. Results showed that the proportions of G4946E generally increased from spring through summer, but then decreased in autumn. Furthermore, the proportions in late autumn were similar to those in early spring of the subsequent year. These results suggest that diamide effectiveness for P. xylostella control in the reference year can be evaluated based on a proportion survey in the prior year.

Cypermethrin resistance and reproductive types in onion thrips, Thrips tabaci (Thysanoptera: Thripidae).

Cypermethrin resistance and reproductive types were examined for T. tabaci strains. Some arrhenotokous and thelytokous strains encoded the sodium channel mutation (T929I) involved in cypermethrin resistance. However, the resistance levels varied to some degree among the strains. A cytochrome P450 inhibitor, piperonyl butoxide, showed different synergistic effects on the strains examined. These results suggest that fundamental and additional levels of cypermethrin resistance in T. tabaci are conferred respectively by reduced sensitivity of the sodium channel and by cytochrome P450-mediated detoxification.

Management of apple orchards to conserve generalist phytoseiid mites suppresses two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae).

To improve the success of integrated pest management (IPM) in apple orchards, we investigated whether generalist phytoseiid mites have suppressed the occurrence of Tetranychus urticae. In Akita Prefecture, northern Japan, in 2012 and 2013, two types of experimental plot were compared. Conservation plots had been managed for the conservation of generalist phytoseiid mites by selective chemical spraying without mowing since 2009. Conventional plots were managed by non-selective chemical spraying with regular mowing. The conservation plots had significantly fewer T. urticae adult females per tree in both years. Two species of generalist phytoseiid mites-Typhlodromus vulgaris and Amblyseius tsugawai-were continuously present in the conservation plots, with only a few T. urticae. The conservation plots had significantly more A. tsugawai adult females in the undergrowth in both years, and significantly more T. vulgaris adult females on apple leaves in 2012. Typhlodromus vulgaris was continuously present in the conservation plots but was scarce from late May to early August in the conventional plots. In the presence of T. vulgaris, low numbers of T. urticae did not increase on apple leaves. These results indicate that the generalist phytoseiid mites serve as important biological control agents in IPM in apple orchards.

Spinosad resistance of melon thrips, Thrips palmi, is conferred by G275E mutation in α6 subunit of nicotinic acetylcholine receptor and cytochrome P450 detoxification.

To examine the resistance mechanisms of Thrips palmi against spinosad, we cloned partial nucleotide sequences of the nicotinic acetylcholine receptor α6 subunit (TPα6) gene from susceptible (OK) and resistant (TS1 and TS5) strains and compared the deduced amino acid sequences among the three strains. The OK, TS1, and TS5 strains respectively showed LC50 values of 3.4mg/L, 2838.5mg/L, and 6655.5mg/L. The deduced amino acid sequence of TPα6 gene showed 96% identity with that of Frankliniella occidentalis. Comparison of the deduced amino acid sequences of TPα6 gene among the three strains showed that the TS1 and TS5 strains had a resistant amino acid, Glu, at amino acid position 275. On the other hand, a susceptible amino acid, Gly, was encoded at the corresponding amino acid position for the OK strain. The synergist, piperonyl butoxide, respectively caused 1.1-fold , 5.8-fold , and 9.0-fold decreases in the resistance ratios of the OK, TS1, and TS5 strains. These results suggest that spinosad resistance of T. palmi is conferred by reduced sensitivity of TPα6 and cytochrome P450-mediated detoxification.

Duplication of acetylcholinesterase gene in diamondback moth strains with different sensitivities to acephate.

This study examined the acetylcholinesterase 1 gene (AChE1) in Plutella xylostella strains with different sensitivities to acephate. Multiple haplotypes of the gene were found in the field-collected strains including distinct haplotypes carrying one or both previously reported mutations (A298S and G324A). Moreover, sequencing results indicated the presence of duplicated copies of the gene in the field-collected strains. No correlation was found between copy numbers of AChE1 and levels of resistance to acephate suggesting that extensive AChE1 duplication is not a major resistance factor at least in some P. xylostella strains. Proportions of the A298S and G324A mutations showed no correlation with levels of resistance to acephate. This suggests that acephate resistance of P. xylostella is complex and cannot be evaluated based on the AChE1 copy number or proportions of the resistance mutations alone.

Population survey of phytoseiid mites and spider mites on peach leaves and wild plants in Japanese peach orchard.

A population survey of phytoseiid mites and spider mites was conducted on peach leaves and wild plants in Japanese peach orchards having different pesticide practices. The phytoseiid mite species composition on peach leaves and wild plants, as estimated using quantitative sequencing, changed during the survey period. Moreover, it varied among study sites. The phytoseiid mite species compositions were similar between peach leaves and some wild plants, such as Veronica persica, Paederia foetida, Persicaria longiseta, and Oxalis corniculata with larger quantities of phytoseiid mites, especially after mid-summer. A PCR-based method to detect the ribosomal ITS sequences of Tetranychus kanzawai and Panonychus mori from phytoseiid mites was developed. Results showed that Euseius sojaensis (specialized pollen feeder/generalist predator) uses both spider mites as prey in the field.

Genomic analyses of sodium channel α-subunit genes from strains of melon thrips, Thrips palmi, with different sensitivities to cypermethrin.

We examined the genomic organization of the sodium channel α-subunit gene in two strains of melon thrips, Thrips palmi, having differing sensitivity to cypermethrin. The nucleotide sequences of the strains included 18 or 16 putative exons which covered the entire coding region of the gene producing 2039 amino acid residues. Deduced amino acid sequences of both strains showed 80% homology with those of Periplaneta americana and Cimex lectularius. Comparison of deduced amino acid sequences of both strains showed no consistent amino acid difference. In addition to the previously reported resistant amino acid (Ile) at the T929I site, both strains encoded another resistant amino acids at two positions which are involved in pyrethroid resistance in other arthropods. These amino acids might also involve in the basal levels of resistance to pyrethroids of both strains.