Tadashi Miyata

RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin.

We have previously reported that a cytochrome P450, CYP6BG1, from Plutella xylostella was found to be overexpressed in 4th instars of a permethrin resistant strain and inducible in the susceptible counterpart. The findings suggested potential involvement of CYP6BG1 in permethrin resistance, hence warranted a functional analysis. To assess the functional link of the gene to permethrin resistance, we adopted RNA interference-mediated gene silencing (RNAi) by dsRNA droplet feeding. Here, real time PCR analyses show that oral delivery of dsRNA can efficiently reduce the expression of CYP6BG1. Knockdown of CYP6BG1 transcript was evident in midgut and larval tissues enclosed in carcass. As a consequence of knockdown, a significant reduction in resistance of larvae fed CYP6BG1 dsRNA was observed after 24 and 48h of exposure to permethrin. In addition, CYP6BG1 dsRNA feeding to larvae led to reduced total P450 activities of microsomal preparations toward model substrates p-nitroanisole and benzyloxyresorufin. These results indicate that the overexpressed CYP6BG1 participate in enhanced metabolism of permethrin, thereby, resistance. The knockdown of a non-overexpressed P450, CYP6BF1v4, from the same resistant P. xylostella strain did not lead to changes in the level of resistance to permethrin, supporting further the specific involvement of CYP6BG1 in the resistance.

Synergism of enzyme inhibitors and mechanisms of insecticide resistance in Bemisia tabaci (Gennadius) (Hom., Aleyrodidae)

Abstract:  Synergism of enzyme inhibitors and mechanisms of insecticide resistance in Bemisia tabaci (Gennadius) (Hom., Aleyrodidae), collected from the commercial cruciferous (Brassica oleracea var. italica L.) vegetable fields in Shangjie, Minhou County, Fujian, China (Sj), were studied by using the dry film method and biochemical analysis. In comparison with the insecticide‐susceptible insectarium population of B. tabaci, the resistance ratios in the field population were determined to be 29.3 for methamidophos, 21.8 for chlorpyrifos, 2.2 for phoxim, 72.4 for fenvalerate, 9.4 for avermectin, 5.5 for emamectin benzoate, 1.8 for spinosad, 11.6 for fipronil and 8.0 for imidacloprid. The field population of B. tabaci displayed significantly low acetylcholinesterase (AChE) activity, but high AChE insensitivity to methamidophos and dichlorvos, as well as high carboxylesterase (CarE) activity. Piperonyl butoxide (PB), triphenyl phosphate (TPP) and diethyl maleate (DEM) showed low synergistic effects on the susceptibility to the nine insecticides in the insetarium population of B. tabaci. On the other hand, in the field population of B. tabaci, high synergism ratios to the nine insecticides with PB, to methamidophos, chlorpyrifos, phoxim, fenvalerate and imidacloprid with TPP, and to methamidophos and avermectin with DEM were found. Significant inhibition of AChE activity by PB and of glutathione‐S‐transferase (GST) activity by DEM in the insectarium and field population, of CarE activity by PB, TPP and DEM in the insectarium populations, and of CarE activity by PB and DEM in the field population were found in vivo. The results suggest that the resistance to organophosphates in B. tabaci was associated partly with AChE insensitivity. Oxidative degradation was believed to be the major mechanism of insecticide resistance in B. tabaci found in Sj. Hydrolytic reactions might also be partially involved in the resistance in some cases. These findings indicate that more than one target besides the traditional ones for PB, TPP or DEM might exist in B. tabaci. This might provide a plausible explanation for the improvement in insecticide toxicity by the three enzyme inhibitors. Especially, the highest synergism of PB on the nine insecticides could now be explained by PBs multiple attack on the activity of Car or AChE in B. tabaci.

Variations in sperm number in relation to larval crowding and spermatophore size in the armyworm, Pseudaletia separata

1. Number of sperm and its relationship with larval rearing density were investigated in the armyworm Pseudaletia separata. Males that emerged from crowded larvae produced significantly more apyrene sperm than those from solitary larvae (375 700 ± 116 600 and 290 300 ± 99 600 at a mating with a 3‐day old virgin, respectively), with no significant difference in number of eupyrene sperm between the two types being observed.

Cuticular penetration of S-fenvalerate in fenvalerate-resistant and susceptible strains of the diamondback moth, Plutella xylostella (L.)

Abstract The cuticular penetration of fenvalerate was investigated in two fenvalerate-resistant (OKR 84 -FR and KAR 84 -FR) and two susceptible (OKR 84 and KAR 84 ) strains of the diamondback moth, Plutella xylostella (L.). Cuticular penetration of S -[ 14 C]fenvalerate into larvae of the resistant stains was slower than that of the susceptible strains. The resistant strains showed lower amounts of internal accumulation of S -[ 14 C]fenvalerate. However, the susceptible strains seemed to show higher rates of S -[ 14 C]fenvalerate excretion than the resistant strains. Reduced cuticular penetration of S -[ 14 C]fenvalerate was considered to be an important mechanism of fenvalerate resistance in the diamondback moth.

Mechanisms of resistance to spinosad in the western flower thrip, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)

Cross‐resistance, resistance mechanisms, and mode of inheritance of spinosad resistance were studied in the western flower thrip, Frankliniella occidentalis (Pergande). Spinosad (naturalyte insecticide) showed low cross‐resistance to prothiophos (organophosphorus insecticide) and chlorphenapyr (respiratory inhibitor) showed some cross‐resistance to thiocyclam (nereistoxin). The synergists PBO (piperonyl butoxide), DEM (diethyl maleate), and DEF (s, s, s‐tributyl phosphorotrithioate) did not show any synergism on the toxicity of spinosad in the resistant strain (ICS), indicating that metabolic‐mediated detoxification was not responsible for the spinosad resistance, suggesting that spinosad may reduce sensitivity of the target site: the nicotinic acetylcholine receptor and GABA receptor. Following reciprocal crosses, dose‐response lines and dominance ratios indicated that spinosad resistance was incompletely dominant and there were no maternal effects. The results of backcross showed that spinosad resistance did not fit a single‐gene hypothesis, suggesting that resistance was influenced by several genes.

Seasonal Changes of Methamidophos Susceptibility and Biochemical Properties in Plutella xylostella (Lepidoptera: Yponomeutidae) and Its Parasitoid Cotesia plutellae (Hymenoptera: Braconidae)

Abstract Methamidophos resistance and acetylcholinesterase (AChE) insensitivity to methamidophos, dichlorvos, and carbofuran were determined in the field populations of Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) and its parasitoid Cotesia plutellae Kurdjumov (Hymenoptera: Braconidae) collected from the corresponding hosts between October 1998 and December 2003 in Fuzhou and Minhou, Fijian, China. Resistance levels to methamidophos and AChE insensitivity to the three insecticides in the two species of insects were high during autumn and spring and low during summer. Resistance to methamidophos was 15.3- and 12.6-fold higher in resistant F0 parents of P. xylostella and C. plutellae than in their susceptible F11 progeny, respectively. The bimolecular rate constant (ki) values of AChE to methamidophos, dichlorvos, and carbofuran were 4.6-, 6.3-, and 7.7-fold higher in F11 progeny of P. xylostella, and 3.7-, 4.5-, and 3.7-fold higher in F11 progeny of C. plutellae than those in their F0 parents, respectively. Compared with susceptible F11 progeny, the resistance ratios for methamidophos were 4.2–29.8 and 3.8–13.1 in 21 field populations of P. xylostella and C. plutellae, respectively. The ki values of AChE to methamidophos, dichlorvos, and carbofuran were 2.0–21.6-, 3.6–9.5-, and 2.6–9.2-fold higher in F11 progeny of P. xylostella, and 1.8–7.6-, 1.9–4.6-, and 2.2–7.6-fold higher in F11 progeny of C. plutellae than those in 21 field populations, respectively. Significant correlative variations of methamidophos resistance as well as significant correlative variations of ki values of AChE to insecticides between the two species of insects also were found in space and time. The ki values of AChE to insecticides in C. plutellae were far higher than those in P. xylostella. There were no obvious differences in the Km and Vmax of AChE between F0 parents and F11 progeny of P. xylostella and C. plutellae, respectively. But carboxylesterase activity was 1.6-fold higher in F0 parents of C. plutellae than in F11 progeny, and glutathione S-transferase activity was 1.5-fold higher in F0 parents of P. xylostella than in F11 progeny. The results suggested that the AChE insensitivity to insecticides might play the most important role in methamidophos resistance in the two species of insects. From these results, a spatial and temporal correlative evolution of methamidophos resistance and insensitive AChE was found to exist between P. xylostella and C. plutellae.

Eupyrene and apyrene sperm and their numerical fluctuations inside the female reproductive tract of the armyworm, Pseudaletia separata

Abstract At 1 h after copulation both eupyrene (average length = 1.4 ± 0.1 mm) and apyrene (average length = 0.5 ± 0.1 mm) sperm were found in the sperm storage organ, spermatheca, and at 6 h after copulation, almost all of the sperm (293,900 ± 122,100, with 92.5 ± 2.9% being apyrene sperm) finished their travel from the spermatophore to spermatheca. The number of sperm inside the female reproductive tract did not decrease until 19 h after copulation. The spermatheca between 9 and 15 h after copulation was the best time and place for counting the sperm a male transferred to a female. Unlike the moderate decrease in the number of eupyrene sperm after copulation, the number of apyrene sperm decreased sharply within 3 days after copulation, especially within the first 24 h. This decrease in apyrene sperm correlates with the females remating pattern, suggesting that the number of apyrene sperm may have some influence on female remating.

Identification and characterization of a cytochrome P450 CYP6CX1 putatively associated with insecticide resistance in Bemisia tabaci

Abstract  The novel full length of cytochrome P450 gene has been isolated in insecticide‐resistant (named CYP6CX1v1) and ‐susceptible (named CYP6CX1v2) Bemisia tabaci, which was identified as B biotype, in Shangjie, Fujian, China (Sj). CYP6CX1 (1 940 bp contained a 1 557 bp open reading frame) included conserved domains common to CYP6 members, such as heme‐binding motif PFGEGPRFCIA, putative “meander”‐binding sequence ETLR and PERF in helix‐K, oxygen‐binding motif AGLDPV and conserved sequence PEKFNP near the carboxyl end. There were four different replacements of amino acid residues between R and S B. tabaci (Thr300 Ala, Thr354Pro, Arg486His and Ile503Thr), among which the substitution Ile503Thr was located in the substrate recognition sites region. The mRNA transcription level of CYP6CX1v1 was 2.38‐fold as high as that of CYP6CX1v2. The results indicated that the CYP6CX1 from the B biotype B. tabaci in Sj was one of the CYP6 members, and enhanced CYP6CX1 expression and substitute of amino acid residues might be involved in the resistance mechanisms in field B. tabaci.

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.

Laboratory selection for resistance with phenthoate and fenvalerate in the diamond-back moth, Plutella xylostella L. (Lepidoptera: Yponomeutidae)

Abstract Diamond-back moth was tested for resistance to phenthoate and fenvalerate. The Okinawa (OKR) strain had decreased in insecticide resistance to all insecticides tested, except methomyl, and this species and the Osaka susceptible (OSS) strain of the diamond-back moth were selected for resistance to phenthoate utilizing the spraying method. High levels of resistance to phenthoate could be obtained after eight selections during nine generations. At LD 50 and LD 95 levels, the OKR-selected (OKR-R) strain exhibited 172- and 287-fold resistance to phenthoate, while the OSS-selected (OSS-R) strain exhibited 194- and 289-fold resistance, respectively. No significant difference in the rate of development of phenthoate resistance between the OKR-R and OSS-R strains was observed. On the other hand, selection for resistance with fenvalerate in the OKR strain was limited: only slight resistance could be obtained after 16 selection treatments for 23 generations.