Cell culture of the rice brown planthopper,Nilaparvata lugens Stål (Hemiptera: Delphacidae)

The rice brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive insect pests of rice in Asia. Although resistant rice varieties can be effective in managing planthopper populations, BPH has repeatedly been able to overcome resistant rice varieties. It is possible that BPH adaptation to resistant rice varieties may be related to its endosymbionts. We investigated methods for culturing BPH cells in order to study in-depth interactions between rice, BPH, and its endosymbionts. In this study, we tested EX-CELL? 405, EX-CELL? 420, Mitsuhashi and Maramorosch’s medium, and Kimura’s medium, for in vitro culture of BPH cells. Only Kimura’s medium was found to be suitable for BPH cell culture, and BPH embryos around blastokinetic stage were the best source for BPH cell culture. Cells from BPH embryonic tissues adhered to the plate substrate, and the migration of cells was observed within 24 h in primary culture. After 3 mo of subculture, various types of BPH cells were successfully maintained in the Kimura’s medium.     

Fork head transcription factor is required for ovarian mature in the brown planthopper, Nilaparvata lugens (Stål)

Background

The NCI-60 is a collection of tumor cell lines derived from a variety of human adult cancer tissue types and is commonly used for genetic analysis and screening of potential chemotherapeutic agents. We wanted to understand the contributions of specific mechanisms of genomic instability to the etiology of cancers represented by the NCI-60.

Results

We screened the NCI-60 for dysregulated homologous recombination by using the gene cluster instability (GCI) assay we pioneered, and for defects in base excision repair by sensitivity to 5-hydroxymethyl-2'-deoxyuridine (hmdUrd). We identified subsets of the NCI-60 lines that either displayed the characteristic molecular signature of GCI or were sensitive to hmdUrd. With the exception of the NCI-H23 lung cancer line, these phenotypes were not found to overlap. None of the lines examined in either subset exhibited significant changes in the frequency of sister chromatid exchanges (SCE), neither did any of the lines in either subset exhibit microsatellite instability (MSI) indicative of defects in DNA mismatch repair.

Conclusions

Gene cluster instability, sensitivity to hmdUrd and sister chromatid exchange are mechanistically distinct phenomena. Genomic instability in the NCI-60 appears to involve only one mechanism of instability for each individual cell line.     

Cultivar and insecticide applications affect the physiological development of the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Nilaparvata lugens (St?l) (Hemiptera:Delphacidae) is a serious migratory pest of rice in Asia. Pest resurgence often occurs because of insecticide overuse. Using both susceptible (TN1) and moderately resistant (Xieyou 963) cultivars, we studied the effect of foliar insecticides on the percentage of brachypterous adults, female reproductive rate, and crude fat and soluble sugar contents in third- and fifth-instar nymphs and adults. The percentage of brachypterous adults and reproductive rate of adult females developed from nymphs that fed on insecticide-treated plants varied significantly with rice cultivar, type of insecticide, and its concentration. Feeding on susceptible plants increased the percentage of brachypterous adults and reproductive rate of adult females. Also, deltamethrin increased brachypterous production relative to imidacloprid and triazophos. The highest reproductive rate was on plants treated with triazophos. All insecticide treatments in both cultivars resulted in increase of soluble sugar contents in third- and fifth-instar nymphs and adults developed from nymphs feeding on insecticide-treated rice plants. This effect was stronger on the susceptible cultivar. Changes of crude fat content after N. lugens feeding on insecticide-treated plants were related to its feeding duration. Crude fat content in adult developed from nymphs feeding on treated plants was significantly higher that on control plants. These studies showed that plant and insecticide influences on physiological ecology of this planthopper will influence its population dynamics under commercial production of rice in Asia.     

A rigorous population model for the brown planthopper,Nilaparvata lugens Stål (Homoptera: Delphacidae)

Population Ecology - A rigorous mathematical model is formulated for describing population growth of the brown planthopper (Nilaparvata lugens Stål, Homoptera), a major rice pest in Asia. The...     

Antifeedant and insecticidal effects of mandelic acid on the brown planthopper Nilaparvata lugens Stål

To study the effects of mandelic acid (MA) on the brown planthopper (BPH), Nilaparvata lugens, the survival rate and behaviour of BPH fed on an artificial diet with different dosages of MA was observed. The survival rate of BPH decreased with the increase of the MA concentration and feeding time. In contrast to the control, the survival rate of BPH 72 h after feeding decreased significantly. Electrical penetration graph (EPG) data indicated that MA absorbed by the rice plant from Kimura B solution significantly affected the feeding behaviour of BPH. At the concentrations of 0.1, 0.5, and 1.0 mg/ml, duration of the phloem ingestion of BPH decreased from 115.34 min (control) to 30.41, 7.63, and 0.36 min, respectively. Periods of xylem ingestion of MA-treated BPH were significantly shorter than those of the control (50.44 min). Moreover, BPH spent more time walking around or being at rest on MA-treated rice plants, as well as in stylet activities. The GST (glutathione S-transferase) activity of BPH increased with the increasing MA concentration, while the GPX (glutathione peroxidases) activity did not change significantly. The results indicate that MA has an antifeedant and insecticidal effect on BPH.     

Molecular mapping and genetic analysis of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene

The brown planthopper (BPH), Nilaparvata lugens St?l, is a serious insect pest of rice (Oryza saliva L.). We have determined the chromosomal location of a BPH resistance gene in rice using SSR and RFLP techniques. A rice line 'B14', derived from the wild rice Oryza latifolia, showed high resistance to BPH. For tagging the resistance gene in 'B14X', an F2 population and a recombinant inbred (RI) population from a cross between Taichung Native 1 and 'B14' were developed and evaluated for BPH resistance. The results showed that a single dominant gene controlled the resistance of 'B14' to BPH. Bulked segregant SSR analysis was employed for identification of DNA markers linked to the resistance gene. From the survey of 302 SSR primer pairs, three SSR (RM335, RM261, RM185) markers linked to the resistance gene were identified. The closest SSR marker RM261 was linked to the resistance gene at a distance of 1.8 cM. Regions surrounding the resistance gene and the SSR markers were examined with additional RFLP markers on chromosome 4 to define the location of the resistance gene. Linkage of RFLP markers C820, R288, C946 with the resistance gene further confirmed its location on the short arm of chromosome 4. Closely linked DNA markers will facilitate selection for resistant lines in breeding programs and provide the basis for map-based cloning of this resistance gene.     

Physiological roles of a yeast-like symbiote in reproduction and embryonic development of the brown planthopper,Nilaparvata lugens Stål

The physiological roles of a yeast-like symbiote in oviposition and embryonic development of its host, the brown planthopper, Nilaparvata lugens Stål, were studied using heat treatment, lysozyme injection and ligation of eggs. The eggs laid by the heat-treated females harboured only a few of the symbiotes, and their symbiote ball through embryonic development was free of symbiotes. The embryos of subsymbiotic eggs could not undergo blastokinesis and dorsal closure, and failed to hatch due to lack of differentiation of the abdominal segments. Electrophoretic profile of the eggs laid by the heat-treated females indicated the absence of several minor proteins which are usually found in the fat body of normal females. A protein (Y) of 131 kD was barely detectable in the heat-treated insects, and could not be found in the ligated eggs in which the symbiote ball was completely separated from the developing germ band. It is suggested that the symbiote supplies its host with proteins for normal embryonic and postembryonic development. The number of yeast-like symbiotes in female adults was reduced after injection with lysozyme solution, and some of the eggs were unable to hatch due to failure in blastokinesis; this was similar to the heat-treated insects. The embryos of ligated eggs could complete segmentation and differentiation normally before 110 h, but the abdominal segments failed to differentiate after dorsal closure, and regressed leaving only the head. Partially ligated eggs harboured some symbiotes and could produce normal larvae. It is concluded that the yeast-like symbiote is significant in abdominal segmentation and differentiation of the planthopper embryo.     

Differential glycosylation produces heterogeneity in elevated esterases associated with insecticide resistance in the brown planthopper Nilaparvata lugens Stål

The major insecticide resistance mechanism in the brown planthopper Nilaparvata lugens involves overproduction of esterases. Esterases purified from a resistant strain appeared as a ladder of bands on isoelectric focussing (IEF) gels from pI 4.7 to 5.0. Two-dimensional electrophoresis showed that isozymes ranged in size from 66 to 68 kDa with those of lower pI being apparently smaller. All isozymes detected by two-dimensional electrophoresis were glycosylated. N-glycosidase A reduced the number of isozymes on IEF to two, with increased pI and an increased molecular weight of 69 kDa. No O-linked glycans were detected. Deglycosylation had no effect on esterase activity, hence glycosylation is not involved in active site conformation. As N-glycosidase F completely deglycosylated the esterases, none of the glycans has an alpha1,3-bound core fucose. Reactivity with the lectins GNA, MAA and DSA, combined with differential cleavage of N-linked glycans with endoglycosidases F1 and F2, indicated that terminally linked mannose is present in high mannose and/or hybrid type glycans and that terminally linked sialic acid and galactose-beta(1-4)-N-acetylglucosamine are present in biantennary complexes. Neuraminidase treatment had the same effect on pI of isozymes as complete deglycosylation. Therefore, the majority of the heterogeneity of elevated esterases on IEF is due to differential attachment of sialic acid to glycans of the two proteins.     

Characterization and functional study of juvenile hormone diol kinase gene in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

The brown planthopper, Nilaparvata lugens (Stål) is an important pest in rice. It has been widely recognized that the juvenile hormone (JH) is regulated by its hydrolase, which includes juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK). In this paper, we cloned the gene of Jhdk and the gene expression at different stages of N. lugens was analysed, and the relationship with Jhe and Jheh was studied after silencing the jhdk gene of N. lugens (Nljhdk) through double-stranded RNA (dsRNA) feeding. We also explored the expression of the three JH hydrolase after indoxacarb treatments. RT-PCR was used to amplify the full length Jhdk cDNA, and the Nljhdk gene was expressed throughout all the development periods tested and showed the lowest level at the 4th instar and the highest in the 5th instar. The expression level of Nljhdk in male adults was higher than that of female adults. Through feeding, dsRNA against Nljhdk successfully knocked down the target gene, which had no significant effect on the expression of the jhe gene of N. lugens (Nljhe), while the expression of Nljheh was upregulated. Indoxacarb could inhibit N. lugens reproduction, and the expression level of Nljhe and Nljhdk increased with the increasing of indoxacarb concentration, but the expression of the jheh gene of N. lugens (Nljheh) was reduced. These studies provide a line of experimental evidence in N. lugens to support that Nljhdk encodes the functional protein involved in JH degradation and further showed the relationship of the three hydrolases and the mechanism of indoxacarb inhibition of the fecundity of N. lugens.     

Insulin receptors regulate the fecundity of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Two InR (insulin receptor) genes have been identified in the Nilaparvata lugens. In this study, we used RNA interference (RNAi) to investigate the role of InR genes in the fecundity of N. lugens. The expression of NLInR1 and NLInR2 genes was simultaneously silenced with mixture of dsInR1 and dsInR2 (dsInRs) injection. Our results showed that larvae RNAi against both NLInR1 and NLInR2 reduced the number of eggs laid by N. lugens and some eggs as well as ovaries were abnormal. In addition, the relative expression of Vg (vitellogenin) and VgR (vitellogenin receptor) was significantly reduced on the 4th and 6th days after insects treated with larvae RNAi reached the adult stage. We also determined the relative expression levels of insulin/insulin-like signaling (IIS) related genes in RNAi-treated larvae and found that the expression levels of Chico (homologous receptor substrate), Akt (protein kinase B), PI3K (phosphoinositide 3-kinase), and PTEN (phosphatase and tensin homolog) genes decreased whereas FOXO (forkhead box O) and GSK3 (glycogen synthase kinase-3) levels increased on the 4th and 6th days after insects reached the adult stage. These results indicate that silencing of NLInR1 and NLInR2 reduces the fecundity of N. lugens through the IIS pathway.     

Development and use of EST-SSR markers for assessing genetic diversity in the brown planthopper (Nilaparvata lugens Stål)

To assess genetic diversity in populations of the brown planthopper (Nilaparvata lugens St?l) (Homoptera: Delphacidae), we have developed and applied microsatellite, or simple sequence repeat (SSR), markers from expressed sequence tags (ESTs). We found that the brown planthopper clusters of ESTs were rich in SSRs with unique frequencies and distributions of SSR motifs. Three hundred and fifty-one EST-SSR markers were developed and yielded clear bands from samples of four brown planthopper populations. High cross-species transferability of these markers was detected in the closely related planthopper N. muiri. The newly developed EST-SSR markers provided sufficient resolution to distinguish within and among biotypes. Analyses based on SSR data revealed host resistance-based genetic differentiation among different brown planthopper populations; the genetic diversity of populations feeding on susceptible rice varieties was lower than that of populations feeding on resistant rice varieties. This is the first large-scale development of brown planthopper SSR markers, which will be useful for future molecular genetics and genomics studies of this serious agricultural pest.     

Humid microenvironment prerequisite for the survival and growth of nymphs of the rice brown planthopper,Nilaparvata lugens (Stål) (Homoptera: Delphacidae)

Nymphs ofNilaparvata lugens were experimentally reared from the 2nd instar in a cage covering part of the leaf sheath of an individual rice plant grown in a Wagner pot. Plants were covered with the cage from the water surface of the pot to 10 cm above the surface (lower cage-group) or from 10 cm to 20 cm above the surface (upper cage-group). Temperatures measured at three different parts of the cage remained fairly constant in both groups at around 25°C (23.7–25.2°C in mean value). In the lower cage-group, relative humidities measured at the three heights in the cage in (76.3–90.5% in mean value) markedly increased with the approach to the water surface. The nymphs of this group, particularly during the molting period, aggregated close to the surface. Eighty-two percent of the released nymphs emerged in this group. Relative humidities measured at three heights of the upper cage-group were 69.5–72.7% in mean value, and all the nymphs in this group died within 3 days after their release although half of them stayed on the rice plants within 6 h after their release. The role of relative humidity as a limiting factor on the range of the microhabitat and the population density ofN. lugens in rice fields was discussed on the basis of the results.     

Relative humidity as an environmental factor determining the microhabitat of the nymphs of the rice brown planthopper,Nilaparvata lugens (Stål) (Homoptera: Delphacidae)

Microhabitat of the nymphs and the adults of the rice brown, planthopper, Nilaparvata lugens is known to be the lower parts of rice plants. In this study, possible environmental factors determining the range of the microhabitat were experimentally analyzed by using the nymphs under laboratory conditions. Thirty individuals of the 1st or the 3rd instar nymphs were released to a potted rice plant covered with a transparent acrylic cylinder. When the top of the cylinder was kept opening (open condition), the temperatures in the cylinder were almost constant, and relative humidities in the cylinder decreased with the increase of the height from the water surface of the pot. In the open condition, most nymphs and all exuviae were found on the basal parts of rice plants where the humidity was more than ca. 90% r.h. When the top of the cylinder was kept closing with parafilm (closed condition), the temperatures in the cylinder were almost constant, and relative humidities in the cylinder were more than 95% r.h. In the closed condition, the nymphs and the exuviae were distributed sparsely to the whole parts of rice plants. In both of the open and the closed conditions, patterns of nymphal distributions on rice plants during the dark regime were the same as those during the light regime under 25±2°C and 16L∶8D. Four different temperatures ranging from 20°C to 35°C did not influence on the microhabitat in the open condition. It was concluded from the results that relative humidity is the important environmental factor to determine the microhabitat of the nymphs of N. lugens which showed to prefer very humid condition more than ca. 90% r.h.     

Proteomic analysis of insecticide triazophos-induced mating-responsive proteins of Nilaparvata lugens Stål (Hemiptera: Delphacidae)

The brown planthopper, Nilaparvata lugens (St?l) (Hemiptera: Delphacidae), is a classic example of a resurgent pest induced by insecticides. It has been demonstrated that triazophos treatment causes an increase in the content of male accessory gland proteins (Acps) that can be transferred to females via mating, influencing female reproduction. However, the mechanism of this type of insecticide-induced Acps in males and the subsequent stimulation of reproduction in females are not well understood. To identify changes in the types of Acps and reproductive proteins in mated females, we conducted a comparative proteomic analysis. Six samples were categorized into four different groups: (1) untreated unmated males compared to treated unmated males (UUM vs TUM); (2) treated unmated males compared to treated mated males (TUM vs TMM); (3) untreated unmated females compared to treated unmated females (UUF vs TUF); (4) treated unmated females compared to treated mated females (TUF vs TMF). Protein expression changes among the four different groups were examined by two-dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Of the 500-600 reproducibly detected protein spots on each gel, 107 protein spots were differentially expressed between the four different groups. Of the 63 proteins identified by LC-MS/MS analysis, 38 were up-regulated and 25 were down-regulated in the four different groups. Some novel proteins related to fecundity were observed including spermatogenesis-associated protein 5, testis development protein NYD-SP6, arginine kinase, actin-5C, vitellogenin, and ovarian serine protease nudel. The elevated expression of novel fecundity proteins in six samples of N. lugens females and males due to exposure to triazophos was confirmed by quantitative real-time PCR (qRT-PCR). The results suggest that these proteins may participate in the reproductive process of N. lugens adult females and males. Our findings fill a gap in understanding the relationship between insecticide-treated males and the stimulated reproduction of N. lugens females.     

Changes in searching responses with temperature ofCyrtorhinus lividipennis reuter (Hemiptera: Miridae) on the eggs of the brown planthopper,Nilaparvata lugens (Stål.) (Homoptera: Delphacidae)

The functional response ofCyrtorhinus lividipennis Reuter attacking the brown planthopper(Nilaparvata lugens (Stål.), BPH) eggs on rice was determined at six constant temperatures of 20°C, 23°C, 26°C, 29°C, 32°C and 35°C. Rogers’ (1972) random predator equations were fitted to the data for each temperature separately. The equation model adequately described a Holling’s Type II functional response forC. lividipennis and produced biologically realistic estimates of attack rates and handling times at five experimental temperatures between 20°C and 32°C. However, at 35°C, a negative handling time(Th = - 0.0029) was produced. The effect of temperature was incorporated in to Rogers’ equation by making the attack rate(a) and handling time(Th) functions of temperature within the range 20°C to 32°C. The attack rate and handling time were fitted as hyperbola and reciprocal hyperbola functions of temperature respectively. The temperature incorporating fourparameter composite model fitted the combined data as well as did separate Rogers’ equations based on the parameter estimates.     

Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown planthopper (Nilaparvata lugens Stål) in transgenic rice

Proteinase inhibitors are widely distributed in animals, plants and microorganisms and their roles in plants are associated with defense against pests. The utilization of proteinase inhibitors for crop protection has been actively investigated with a variety of proteinase inhibitors. Soybean Kunitz trypsin inhibitor (SKTI), one of the major seed storage protein, is synthesized for a short period during seed development. To investigate the role of SKTI in a plant's defense system against insect predation, a recombinant plasmid containing the full-length cDNA of SKTI under control of the CaMV 35S promoter was introduced into rice protoplasts by using the PEG direct gene transfer method and a large number of transgenic rice plants were regenerated. The integration, expression, and inheritance of this gene was demonstrated in R1 and R2 generations by Southern, northern, and western analyses. Accumulation levels (0.05–2.5% of soluble proteins) of SKTI protein were detected in R1 and R2 plants. Bioassay with R1 and R2 transgenic plants revealed that transgenic plants are more resistant to destructive insect pest of rice, brown planthopper (Nilaparvata lugens Stål), than the control plants. Thus, introduction of SKTI into rice plants can be used to control insect pests.     

Marker-assisted pyramiding of brown planthopper (Nilaparvata lugens Stål) resistance genes Bph1 and Bph2 on rice chromosome 12

Brown planthopper (BPH) (Nilaparvata lugens St?l) is a significant insect pest of rice (Oryza sativa L.). We constructed a gene-pyramided japonica line, in which two BPH resistance genes Bph1 and Bph2 on the long arm of chromosome 12 independently derived from two indica resistance lines were combined through the recombinant selection. The gene-pyramiding was achieved based on the previously constructed high-resolution linkage maps of the two genes. Two co-dominant and four dominant PCR-based markers flanking the loci were used to select for a homozygous recombinant line in a segregating population that was derived from a cross between the parental homozygous single-gene introgression lines. BPH bioassay showed that the resistance level of the pyramided line was equivalent to that of the Bph1-single introgression line, which showed a higher level of resistance than the Bph2-single introgression line. The pyramid line should provide a useful experimental means for studying the fine structure of the chromosomal region covering these two major BPH resistance genes.     

Effects of rice potassium level on the fecundity and expression of the vitellogenin gene of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Potassium (K) is a key component of plant nutrition, significantly influencing crop growth. Levels of this nutrient in plants can also influence a number of pest infestations. The brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is an important pest of rice in Asia. In this study, we examined K contents in rice grown in hydroponic solution, and its relationship to the fecundity and expression of the vitellogenin (Nlvg) gene of N. lugens which was reared on the rice. Our findings indicated that K contents in rice increased with the increasing K concentration within the hydroponic solution, but reduced at the highest K concentration (160 mg/L). The number of eggs laid by N. lugens which was reared on the rice varied significantly with K concentration, and increased by 0.12 and 0.22 fold under 20 mg/L and 160 mg/L K level compared to that of the control (40 mg/L), decreasing by 0.57 fold under 0 mg/L K. Nlvg mRNA expression increased 1.17 and 1.94 fold in individuals which were reared on rice grown in 20 mg/L and 160 mg/L K level, compared to that of the control before mating; and by 3.36 and 2.97 after mating, respectively. However, Nlvg mRNA expression fold decreased by 0.99 under 0 mg/L K level before mating and 0.91 after mating. The variation of eggs may be attributed to the change of Nlvg mRNA expression, because there was a positive correlation between the eggs and Nlvg mRNA expression. These results demonstrated low (20 mg/L) and highest K levels (160 mg/L) in hydroponic solution showed the lower K level in plants than the control, which facilitated the fecundity of N. lugens. The study of the effects of K levels on the fecundity should have significance for insect control.