Rice planting systems, global warming and outbreaks of Nilaparvata lugens (Stål)

Brown Planthopper (BPH, Nilaparvata lugens (St?l)) is one of the most serious pests of rice in both temperate and tropical regions of East and South Asia and has become especially problematic over the past few years. In order to analyze the effect of the change of rice cropping system on the population dynamics of BPH, field surveys of the occurrence and distribution of BPH were performed and other relevant data, including light trap data and ovary dissection data were collected in nearly 40 Chinese counties encompassing six provinces (or municipalities), including Hainan, Guangxi, Anhui, Shanghai, Fujian and Guangdong from April to October in 2007.The mixed planting areas of single- and double-cropping rice in China include Hubei, South and Central Anhui, North Hunan, and North Jiangxi. In these areas, double-cropping rice has now been greatly reduced and single-cropping rice has been rapidly increasing since 1997. The surveys revealed that when the immigration peak of BPH occurred in June and July, the single-cropping rice was at the tillering to booting stage and fit for BPH, but early rice had already matured and most of late rice had not yet been transplanted. BPH immigrants from southern rice areas prefer to inhabit and breed in single-cropping rice paddies. Moreover, farming activities between early rice and late rice interrupted the continuous growth of BPH populations in double-cropping rice paddies. As a result, in comparison with data collected 30 years ago, the spatiotemporal dynamics and migration patterns of BPH have dramatically changed in the lower-middle reaches of the Yangtze River. In the mixed planting areas, due to their high suitability, the BPH population in single-cropping rice grew so quickly that it caused serious local damage and there was mass emigration of macropterous progeny to the Yangtze River Delta in late August and early September.Global warming may also affect BPH populations, where results suggest steadily warmer autumns have occurred from the 1990s on, with such conditions gradually the norm. The combination of 'cooler summer' and 'warmer autumn' are conditions known to promote outbreaks of BPH in the lower-middle reaches of the Yangtze River. Immigrant BPH arrivals in late August and September now cause serious damage to late-maturing mid-season rice and late rice in the lower-middle reaches of the Yangtze River.     

Oviposition Behavior of the Rice Brown Planthopper, Nilaparvata lugens (Stål), and Its Electronic Monitoring

Oviposition behavior of Nilaparvata lugens (Stål) (Homoptera: Delphacidae) on rice, Oryzae sativa L., was monitored using a videocamera and an AC (alternating-current) electronic measuring system. Oviposition by N. lugens always took place during stylet penetration into the plant. After penetration of the ovipositor, there were three specific ovipositional sequences observed; sawing insertion of the ovipositor, release of an egg in the plant, and partial or complete withdrawal of the ovipositor from the plant. Distinctive waveforms were recorded in correlating with these events. From a sequence of waveform patterns, it was possible to determine the number of eggs and egg masses laid inside the plant tissue and the duration of oviposition behavior. Waveform analysis revealed that N. lugens laid an egg mass consisting of on average 5.7 eggs. This occurred 4.3 min after a period of about 6.4 min of stylet penetration, and this sequence was repeated several times in 24 h. Electrical and/or tethering effects on oviposition appear to be negligible within 24 h of recording.     

Are outbreaks of Nilaparvata lugens (Stål) associated with global warming?

Outbreaks of the Nilaparvata lugens (St?l), have occurred frequently in China during the past few years, resulting in a broad and significant reduction in rice yield. N. lugens immigrate into China each spring from Southeast Asia, and the Guangxi Zhuang Autonomous Region is the first area affected. Light trap catches for the early season period (March-June) in Guangxi for the past 30 yr have been analyzed, and the catch sequences for five observation stations (Longzhou, Hepu, Yongning Yongfu, and Quanzhou) were studied in detail. It was found that during the past 10 yr the first appearance of N. lugens at light traps occurs earlier, there is a higher frequency of days with large light-trap catches, and catches in southern Guangxi are larger. Recently light-trap catches have also increased in northern Guangxi. It is concluded that the increasing number of immigrants from overseas is one of the primary reasons for the increase in N. lugens outbreaks in the past 10 yr. Global warming, and specifically winter temperature increases, appears to be for a factor accelerating outbreaks of N. lugens in Asia.     

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.     

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.     

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.     

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.     

Nutrient uptake of rice roots in response to infestation of Nilaparvata lugens (Stål) (Homoptera: Delphacidae)

This study investigated the uptake of three macronutrients, including nitrogen (N), phosphorus (P), and potassium (K), by rice roots in response to different infestation levels of Nilaparvata lugens (St?l) (Homoptera: Delphacidae). Hydroponics experiments were conducted on the rice variety 'Zhendao 2' (moderately resistant to Tryporyza incertulas, Lepidoptera: Pyralidae) and the variety 'Xiushui 63' (susceptible to N. lugens). In 'Zhendao 2', N. lugens infestation significantly influenced the uptake of P and K but not N, with P and K uptake decreasing as the duration of N. lugens infestation increased. In 'Xiushui 63', N. lugens infestation influenced N, P, and K uptake to a different degree, depending on the infestation level, in which infestation for 2, 4, 6, and 8 d at a density of 60 nymphs did not affect N uptake, but such infestation levels significantly influenced the uptake of P and K. After the removal of N. lugens from rice plants, the N uptake recovered from infestation faster than that for P and K in the variety 'Zhendao 2', whereas the recovery rate of K uptake was faster than that for N and P in the variety 'Xiushui 63'. The recovery rate of the nutrient uptake was negatively correlated to the density and duration of infestation. The experimental results demonstrated that N, P, and K uptake of rice roots were largely not influenced by N. lugens infestation when the pest density was controlled below 15 nymphs per hill. This infestation level was in agreement with the proposed economic thresholds for control measures against the N. lugens infestation on rice plants.     

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...     

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.     

Biochemical mechanism of rice nitrogen promoting the resistance development of Nilaparvata lugens (Stål) to buprofezin

Overusing nitrogen fertilizer not only pollute farmlands, but also make Nilaparvata lugens (Stål) less susceptible to pesticides. The current study investigated the relationship between nitrogen fertilizer application and buprofenzin resistance in N. lugens in rice by conducting a series of field efficacy tests and laboratory bioassays. The results showed that the efficacy of buprofezin decreased significantly with high level of nitrogen fertilizer application, compared to those with low or no nitrogen application in rice fields. In the laboratory, the resistance ratios of N. lugens to buprofezin, assayed at three concentrations of nitrogen (0, 1.0, 3.0 mmol/L), have developed to 14.05-, 18.40- and 25.81-fold after 20 generations of resistance selection, respectively. Meanwhile, the resistance ratios of N. lugens without buprofezin treated were 1.00-, 3.30- and 4.67-fold after 20 generations as the nitrogen concentrations increased. The enzymatic assays revealed that the activities of P450 and esterase increased significantly after resistance selection with buprofezin and nitrogen, especially on the esterase activity in N. lugens under the condition of nitrogen treatment. These results suggested that nitrogen fertilizer application contributed to the resistance development in N. lugens to buprofezin by enhancing the activity of esterase.     

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.     

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.     

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.     

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.     

Distinct Expression Profiles and Different Functions of Odorant Binding Proteins in Nilaparvata lugens St?l

Background

Odorant binding proteins (OBPs) play important roles in insect olfaction. The brown planthopper (BPH), Nilaparvata lugens Stål (Delphacidae, Auchenorrhyncha, Hemiptera) is one of the most important rice pests. Its monophagy (only feeding on rice), wing form (long and short wing) variation, and annual long distance migration (seeking for rice plants of high nutrition) imply that the olfaction would play a central role in BPH behavior. However, the olfaction related proteins have not been characterized in this insect.

Methodology/Principal Findings

Full length cDNA of three OBPs were obtained and distinct expression profiles were revealed regarding to tissue, developmental stage, wing form and gender for the first time for the species. The results provide important clues in functional differentiation of these genes. Binding assays with 41 compounds demonstrated that NlugOBP3 had markedly higher binding ability and wider binding spectrum than the other two OBPs. Terpenes and Ketones displayed higher binding while Alkanes showed no binding to the three OBPs. Focused on NlugOBP3, RNA interference experiments showed that NlugOBP3 not only involved in nymph olfaction on rice seedlings, but also had non-olfactory functions, as it was closely related to nymph survival.

Conclusions

NlugOBP3 plays important roles in both olfaction and survival of BPH. It may serve as a potential target for developing behavioral disruptant and/or lethal agent in N. lugens.     

Antixenosis,tolerance and genetic analysis of some rice landraces for resistance to Nilaparvata lugens (Stål.)

Twenty-six rice landraces from West Bengal, India were evaluated for antixenosis and tolerance against brown planthopper (BPH) biotype 4 at the Bidhan Chandra Krishi Viswavidyalaya (BCKV), West Bengal. High levels of resistance were observed in six landraces, namely Badshabhog, Gamra, Haldichuri, Janglijata, Kalabhat and Khara. These phenotypically resistant rice landraces including Ptb33 exhibited lowest feeding rate, fecundity, nymphal and adult preference, survival, plant dry weight loss per mg of BPH dry weight produced (PDWL), and higher functional plant loss index (FPLI), more days to wilt and unhatched eggs compared with the susceptible check Swarna. All the landraces were classified into four major clusters at 10 unit distance by the scale of similarity during genetic diversity analysis through 21 gene-linked SSR markers of BPH resistance. Some phenotypically resistant landraces were gathered under the major cluster I indicating their analogous genetic history, while some were grouped with susceptible landraces exhibiting their genetic variation. The resistant landraces can be used as potential donors in the breeding programme for the development of rice varieties with resistance to BPH.     

Construction of a high-resolution linkage map of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene bph2

The brown planthopper (BPH), Nilaparvata lugens Stål, is a significant insect pest of rice (Oryza sa-tiva L.). bph2 is one of the 12 major BPH resistance genes so far identified in several indica cultivars and two wild relatives. We have constructed a high-resolution linkage map as a foundation for map-based cloning of the bph2 locus. An advanced mapping population derived from a cross of ’Tsukushibare’ (a susceptible japonica cultivar) with ’Norin-PL4’ (an authentic bph2-introgression line) was used. Segregation analysis by the mass seedling test showed that bph2 behaved as a single dominant gene. Through bulked segregant analysis and linkage analysis, bph2 was located within a 3.2-cM region containing eight AFLP markers. One marker (KAM4) showed complete co-segregation with bph2, and bph2 was mapped within a 1.0-cM region delimited by KAM3 and KAM5, two flanking markers. KAM4 was converted into a PCR-based sequence-tagged-site (STS) marker and its co-segregation with bph2 was validated.