Heat Stress Impedes Development and Lowers Fecundity of the Brown Planthopper Nilaparvata lugens (St?l)

This study investigated the effects of sub-lethal high temperatures on the development and reproduction of the brown plant hopper Nilaparvata lugens (Stål). When first instar nymphs were exposed at their ULT₅₀ (41.8°C) mean development time to adult was increased in both males and females, from 15.2±0.3 and 18.2±0.3 days respectively in the control to 18.7±0.2 and 19±0.2 days in the treated insects. These differences in development arising from heat stress experienced in the first instar nymph did not persist into the adult stage (adult longevity of 23.5±1.1 and 24.4±1.1 days for treated males and females compared with 25.7±1.0 and 20.6±1.1 days in the control groups), although untreated males lived longer than untreated females. Total mean longevity was increased from 38.8±0.1 to 43.4±1.0 days in treated females, but male longevity was not affected (40.9±0.9 and 42.2±1.1 days respectively). When male and female first instar nymphs were exposed at their ULT₅₀ of 41.8°C and allowed to mate on reaching adult, mean fecundity was reduced from 403.8±13.7 to 128.0±16.6 eggs per female in the treated insects. Following exposure of adult insects at their equivalent ULT₅₀ (42.5°C), the three mating combinations of treated male x treated female, treated male x untreated female, and untreated male x treated female produced 169.3±14.7, 249.6±21.3 and 233.4±17.2 eggs per female respectively, all significantly lower than the control. Exposure of nymphs and adults at their respective ULT₅₀ temperatures also significantly extended the time required for their progeny to complete egg development for all mating combinations compared with control. Overall, sub-lethal heat stress inhibited nymphal development, lowered fecundity and extended egg development time.     

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.     

Analysis of Yeast-Like Symbiote Diversity in the Brown Planthopper (BPH), Nilaparvata lugens Stål, Using a Novel Nested PCR-DGGE Protocol

Yeast-like symbiotes (YLS) are endosymbionts that are intimately associated with the growth, development, reproduction of their host, the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). However, it is unclear how many species of YLS are found within N. lugens, and how they are related to each other. Traditional methods or simple amplification based on 18S rDNA sequence does not reliably identify new species quickly and efficiently. Therefore, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategy was developed in this article to analyze the YLS of brown planthopper using a nested PCR protocol that involved the 18S rDNA gene and the 5.8S–ITS gene using fungal universal primers. The nested PCR protocol was developed as follows: firstly, the 18S rDNA gene, and 5.8S–ITS gene were amplified using fungal universal primers. Subsequently, these products were used as a template in a second PCR with primers ITS1GC–ITS2, ITS1FGC–ITS2, and NFGC-NR, which was suitable for DGGE. Using this highly specific molecular approach, we found several previously detected fungi: Noda, Pichia guilliermondii, Candida sp., and some previously undetected fungi, such as Saccharomycetales sp., Debaryomyces hansenii, and some uncultured fungi. In conclusion, the nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a new tool for uncovering fungal endosymbiont diversity within planthoppers.     

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.     

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...     

The significance of morphometric and fecundity differences between the ‘biotypes' of the Brown Planthopper,Nilaparvata lugens

A morphometric analysis of biotypes 1, 2 and 3 from the International Rice Research Institute, Philippines, reared respectively on rice cultivars TN1, Mudgo and ASD7, showed significant differences, but some overlap between them. When the three biotypes were each reared for a single generation on TN1, morphometric differences were very greatly reduced and the distributions widely overlapped.Biotypes 2 and 3 were significantly less fecund than biotype 1 when reared on their normal host varieties. When all were reared on TN1, biotype 3 showed a somewhat lower fecundity than did 1 and 2, but the difference was less than previously reported.It is concluded that the evidence for the association of morphometric differences with virulence characteristics inN. lugens is not proved. Equally there is no evidence that morphometric data may be used to identify field populations with distinct patterns of virulence.

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La signification des différences morphométriques et de fécondité entre des biotypes deNilaparvata lugens

Résumé Ce que l'on a appelé des biotypes deN. lugens sont des populations caractérisées par différents types de virulence à l'égard de différents cultivars résistants de riz, mis en évidence par des essais variétaux systématiques. Différents chercheurs ont tenté de trouver des caractères morphologiques pour identifier ces biotypes.Nous avons fait une analyse morphométrique des biotypes 1, 2 et 3 de l'International Rice Research Institute (IRRI) Philippines. Quand ils sont élevés sur leur propre varieté — biotype 1 sur TN1, 2 sur Mudgo, 3 sur ASD7 — des différences significatives sont observées, bien qu'il y ait un chevauchement considérable. Quand les 3 populations biotypes sont élevées sur la variété sensible TN1, les différences morphométriques sont réduites et le chevauchement fortement augmenté. Nous pourrions alors conclure qu'une part importante de la différenciation morphométrique est due à des facteurs écologiques et non à des différences génétiques entre les populations. Des chercheurs avaient indiqué des différences de fécondité entre les biotypes de l'IRRI, le biotype 3 étant significativement moins fécond; les résultats publiés son contradictoires. Nos observations suggèrent une certaine diminution de la fécondité pour le biotype 3 élevé sur TN1, mais plus limitée que les autres auteurs ne l'avaient envisagée.Nous en concluons qu'il n'y a pas de véritable preuve pour étayer l'hypothèse que les biotypes deN. lugens sont caractérisés par des paramètres morphométriques génétiquement déterminés. Il est alors fallacieux de suggérer que de tels caractères pourraient être utilisés pour identifier des populations avec différents types de virulence. Nous repoussons aussi l'hypothèse que les biotypes représentent une étape dans le processus de spéciation.

     

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.     

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.     

Antifeedants of Finger Millet,Eleusine coracana GAERTN,Against Brown Planthopper,Nilaparvata lugens (STÅL)

Nine compounds isolated from finger millet as antifeedants for brown planthopper have been identified as known compounds, l-malic acid, isocitric acid, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxy- benzaldehyde, and vitexin, and new constituents, 2-O-[4-hydroxy-(Z and E)-cinnamoyl]glyceric acid and 8-C-β-d-[6″-O-(3-hydroxy-3-methyl)glutaroyl]glucopyranosylapigenin.     

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.     

Virus-Mediated Chemical Changes in Rice Plants Impact the Relationship between Non-Vector Planthopper Nilaparvata lugens St?l and Its Egg Parasitoid Anagrus nilaparvatae Pang et Wang

In order to clarify the impacts of southern rice black-streaked dwarf virus (SRBSDV) infection on rice plants, rice planthoppers and natural enemies, differences in nutrients and volatile secondary metabolites between infected and healthy rice plants were examined. Furthermore, the impacts of virus-mediated changes in plants on the population growth of non-vector brown planthopper (BPH), Nilaparvata lugens, and the selectivity and parasitic capability of planthopper egg parasitoid Anagrus nilaparvatae were studied. The results showed that rice plants had no significant changes in amino acid and soluble sugar contents after SRBSDV infection, and SRBSDV-infected plants had no significant effect on population growth of non-vector BPH. A. nilaparvatae preferred BPH eggs both in infected and healthy rice plants, and tended to parasitize eggs on infected plants, but it had no significant preference for infected plants or healthy plants. GC-MS analysis showed that tridecylic aldehyde occurred only in rice plants infected with SRBSDV, whereas octanal, undecane, methyl salicylate and hexadecane occurred only in healthy rice plants. However, in tests of behavioral responses to these five volatile substances using a Y-tube olfactometer, A. nilaparvatae did not show obvious selectivity between single volatile substances at different concentrations and liquid paraffin in the control group. The parasitic capability of A. nilaparvatae did not differ between SRBSDV-infected plants and healthy plant seedlings. The results suggested that SRBSDV-infected plants have no significant impacts on the non-vector planthopper and its egg parasitoid, A. nilaparvatae.     

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.     

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.     

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.     

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.     

Evidence of Sibling Species in the Brown Planthopper Complex (Nilaparvata lugens) Detected from Short and Long Primer Random Amplified Polymorphic DNA Fingerprints

The inheritance of 31 amplicons from short and long primer RAPD was tested for segregating ratios in two families of the brown planthopper, Nilaparvata lugens, and they were found to be inherited in a simple Mendelian fashion. These markers could now be used in population genetics studies of N. lugens. Ten populations of N. lugens were collected from five locations in Malaysia. Each location had two sympatric populations. Cluster and principal coordinate analyses based on genetic distance along with AMOVA revealed that the rice-infesting populations (with high esterase activity) at five localities clustered together as a group, and Leersia-infesting populations (with low esterase activity) at the same localities formed another distinct cluster. Two amplicons from primers OPD03 (0.65 kb) and peh#6 (1.0 kb) could be considered diagnostic bands, which were fixed in the Leersia-infesting populations. These results represent evidence of a sibling species in the N. lugens complex.     

Pathways of Amino Acid Degradation in Nilaparvata lugens (St?l) with Special Reference to Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH)

Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens.     

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.