Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps

Green rice leafhoppers (Nephotettix cincticeps) secrete watery and coagulable saliva in the feeding process. In our study, the watery salivary secretion was concentrated by ultrafiltration from “fed diet” and subjected to SDS-PAGE. The N-terminal amino acid sequence of the most predominant band at 84 kDa (designated NcSP84) was analyzed by Edman degradation. This sequence was completely consistent with the most abundant protein in the salivary gland extracts, which was separated by two-dimensional gel electrophoresis. Based on the N-terminal amino acid sequence, the complete cDNA of this protein was cloned by 5′- and 3′-RACE using degenerate primers. The deduced NcSP84 contained an open reading frame of 2061 bp encoding a putative 687 amino acids with a putative signal sequence composed of 19 amino acids. The nucleotide and amino acid sequences of NcSP84 did not share statistically significant homology with any sequences in public databases. Motif search predicted that this protein had EF-hands, the most common motif found in Ca²⁺ -binding proteins. As predicted, NcSP84 exhibited Ca²⁺-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca²⁺ tended to decline discretely, depending on the concentration of CaCl₂ with which it was mixed for 1 h before adding SDS buffer. In situ hybridization and immunohistochemistry showed that the NcSP84 gene and gene product were expressed and stored in type III cells, which are the largest lobes in the primary salivary glands. The NcSP84 protein was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca²⁺ ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes.     

The outbreak mechanisms of the green rice leafhopper,Nephotettix cincticeps Uhler,in northern Japan

Population Ecology - Intraspecific regulatory processes keep the population ofNephotettix cincticeps stable at a low density in southern Japan. In northern Japan, however, the yearly population...     

黑尾叶蝉卵黄原蛋白受体基因cDNA的克隆、序列分析及表达模式

【目的】卵黄原蛋白受体(vitellogenin receptor,VgR)属于低密度脂蛋白受体,通过介导内吞作用为发育中的卵母细胞摄取卵黄原蛋白,为胚胎发育提供营养物质,在昆虫生殖过程中发挥关键作用。为研究黑尾叶蝉Nephotettix cincticeps VgR(NcVgR)基因的生理功能及其在生殖中的作用,本研究克隆并解析了NcVgR基因的序列,并对其时空表达进行了研究。【方法】根据黑尾叶蝉转录组数据信息,利用RT-PCR克隆了NcVgR基因,并进行了生物信息学分析;利用实时荧光定量PCR研究了不同发育时期、成虫不同组织NcVgR的表达水平。【结果】NcVgR c DNA序列全长6 676 bp,开放阅读框长度5 568 bp,编码1 855个氨基酸,预测编码蛋白的分子量为206 k D,N端前17个氨基酸为信号肽。序列分析显示,NcVgR具有低密度脂蛋白家族的5个经典保守域,即:配体结合域(ligand-binding domain,LBD)、表皮生长因子前体同源域(EGF-precursor homology domain,EGFP)、O-糖链结构域(O-linked sugar domain,OLSD)、跨膜域(transmembrane domain,TMD)和胞质尾域(cytoplasmic domain)。系统发育分析表明,NcVgR与褐飞虱N.lugens VgR亲缘关系最近。实时荧光定量PCR结果显示,NcVgR转录起始时间为5龄若虫,羽化后转录水平逐渐上升,至羽化后8 d达到峰值,随后下降。有意思的是,随着黑尾叶蝉产卵,NcVgR转录水平再次上升,至羽化后16 d达到最高水平。组织定位结果显示,NcVgR在黑尾叶蝉雌成虫卵巢中特异性高表达,而在雌成虫脂肪体和肠道中微量表达,在雌成虫脑及雄成虫中均未检测到表达。【结论】NcVgR在黑尾叶蝉雌成虫卵巢中特异性表达,并且不同发育时期具有不同的表达量,这为研究黑尾叶蝉的生殖调控机理提供了分子信息。     

Absence of protein polymorphism attributable to insecticide-insensitivity of acetylcholinesterase in the green rice leafhopper, Nephotettix cincticeps

The cDNA sequence of acetylcholinesterase (AChE) from the green rice leafhopper, Nephotettix cincticeps, was amplified, based on conserved peptide sequences of AChEs. A 2.3 kb contiguous sequence, containing an ORF encoding an AChE precursor with 677 amino acid residues was obtained. The deduced protein sequence showed the most similarity to that of AChE in the Colorado potato beetle, having common features in the primary AChE structure. cDNA sequences of individual leafhoppers from an insecticide susceptible strain and the resistant strain Nakagawara, whose methylcarbamate-insensitive AChEs show 10(2) or more I(50) ratio for propoxur, were compared. No fixed inter-strain difference was identified in the protein sequence, though amino acid substitution polymorphism was found at one position in the susceptible strain. Insecticide-insensitivity of leafhopper AChE does not result from changes in the protein primary structure that is encoded by the AChE gene sequence isolated in this study.     

Analytical studies of light trap records in the Hokuriku district,II the green rice leafhopper,Nephotettix cincticeps

Light-trap records on the green rice leafhopper, Nephotettix cincticeps, were dealt with to study its population fluctuations in the Hokuriku district. Crude data were modified for distinguishing years of the low intensity of infestation by the insect from the rest of years. It is then clearly demonstrated that the low intensity of infestation were ordinarily preceded by heavy snowfall, although heavy snowfall could not be regarded as the only factor checking the vigorous multiplication of the populations.     

RNA interference‐mediated knockdown and virus‐induced suppression of Troponin C gene adversely affect the behavior or fitness of the green rice leafhopper,Nephotettix cincticeps

The green rice leafhopper, Nephotettix cincticeps, is a major rice pest in Southeast Asia and Southern China. Novel control strategies must be explored to control the rice pest. Behavior or fitness regulation of insect by modulating the Troponin C (TnC) may be a novel strategy in the comprehensive management of the insect pest. However, characterizations and functions of TnC, especially regarding effect of its RNA interference‐mediated gene knockdown on the behavior or fitness of N. cincticeps remain unknown. Here, we successfully cloned and characterized TnC gene from N. cincticeps (Nc‐TnC). We demonstrated that Nc‐TnC ubiquitously transcribed at all development stages and special tissues in adult insects, with relative higher levels at the adult stage and in the intestinal canal. Microinjection‐ or oral membrane feeding‐based transient knockdown of Nc‐TnC adversely affected the performance or fitness, such as the decreased survival, feeding capacity, weight, and fecundity of N. cincticeps. Furthermore, we revealed that the expression of Nc‐TnC was suppressed by its interaction with rice dwarf virus‐encoded nonstructural protein 10, which ultimately affected detrimentally the corresponding parameters of the performance or fitness of N. cincticeps. In conclusion, our data deepen understanding of Nc‐TnC functions during the development of and viral infection in N. cincticeps. It imply Nc‐TnC may serve as a potential target for N. cincticeps control in future.     

Quantitative evaluation of predation by spiders on the green rice leafhopper,Nephotettix cincticeps Uhler,by a sight-count method

A sight-count method for evaluation of predation by spiders on the green rice leafhopper, Nephotettix cincticeps was proposed and its applicability was tested under natural conditions. The number (n) of leafhoppers preyed on by spiders per rice hill per day was estimated by the formula: (1) where F is the frequency of predation observed per hill:P is given by dividing the time spent feeding on prey by 24 hours; and C refers to the total amount of feeding activity expressed in terms of the activity during the standard time interval. The total number (N) of prey attacked during the specified period can be given as follows: (2) With this method, the role of paddy-inhabiting spiders, Lycosa pseudoannulata, Oedothorax insecticeps, Tetragnatha spp, and Enoplognatha japonica, as predator of N. cincticeps was evaluated with reference to life tables of the prey. The advantages and limitation of the sight-count method were discussed as compared with other methods so far proposed.     

Life tables and population parameters of the green leafhopper,Nephotettix cincticeps (Uhler), in the southwestern district of Japan with special reference to the first generation on foxtail grass

The three year (1988–1990) life tables of Nephotettix cincticeps were constructed, and the daily survival rate and longevity of female adults were estimated by Hokyo and Kiritani's (1967) method for the overwintering and the first-generations on the foxtail grass in Okayama, southwestern Japan. The life tables and the population parameter values estimated were compared with those in the other generations on rice. The FARMCOP suction sampler was employed to survey the population density. The durations of pre-ovarial maturation of female adults of the 1st generation on foxtail grass and rice seedling were similar. Longevity of adults of the overwintering and the first generations which emerged on the wild host was longer than that of the other generations (2nd and 3rd generations) on the rice plants. Fecundity of females decreased successively as the generation proceeded and it became lowest in the final 3rd generation. Only about 3.5 percent of first-instar nymphs of the 1st generation emerged as adults in the fallow field. The survival rate of nymphs on foxtail grass was always lower in comparison with that on rice plants. However, the survival rates of nymphs on foxtail grass and rice seedling were not significantly different from each other under laboratory conditions. In the fields, senescence of foxtail grass occurred in the midst of nymphal period of the 1st generation. The survival rate of nymphs on foxtail grass decreased with the increasing in the nymphal density. Abundance of spiders during the 1st generation was higher than that in the early stage of rice plants.     

Life table analysis of the green rice leafhopper,Nephotettix virescens (distant) (Hemiptera: Cicadellidae), and efficient vector of rice tungro disease in asynchronous rice fields in Indonesia

Population dynamics of Nephotettix virescens was studied in 17 paddy fields transplanted at intervals of about 1 month in 1988–1990. The adult density was highest either in the immigrant or the 1st generation and sharply decreased to the 2nd generation. The survival rate of the 1st generation was lowest in the transition season when areal population density increased. Key factor analysis revealed that the nymphal and adult mortality of the 1st generation (kn) was the principal source of population fluctuations. No significant correaltion was found between kn and natural enemy density, natural enemy density/healthy egg density, or the precipitation during the nymphal period. On these bases adult emigration was suspected to be the key factor. Areal population build-up of N. virescens in the transition season was considered to occur as a result of increasing immigration to young stages of rice.     

Numerous peptidoglycan recognition protein genes expressed in the bacteriome of the green rice leafhopper Nephotettix cincticeps (Hemiptera,Cicadellidae)

Applied Entomology and Zoology - Peptidoglycan recognition proteins (PGRPs) contribute to the immune response of vertebrates and higher invertebrates. Animals have a varying number of PGRP genes: 4...     

High mortality caused by high dose of dsRNA in the green rice leafhopper Nephotettix cincticeps (Hemiptera: Cicadellidae)

RNA interference (RNAi) is a useful tool for gene functional studies in non-model insects and pest insects. Establishment of experimental conditions for RNAi, which differ from insect to insect, is important for evaluating the effect of dsRNA injection of relevant genes. When injecting dsRNA into the green rice leafhopper Nephotettix cincticeps (Uhler), high mortality was observed. Therefore, the adverse effects of injection of dsRNA on leafhopper development were examined to assess the suitable conditions for RNAi in this species. Injection manipulation by using a glass capillary did not affect leafhopper survival but delayed the molt of the corresponding instar. High mortality was observed when large amounts of dsRNA were administered. This adverse effect of dsRNA was examined in 2 genes, exogenous EGFP gene and endogenous peptidoglycan recognition protein gene (NcPGRP12). Injection of a high dose (60 ng/insect) caused high mortality in all stages tested: 4th instar, 5th instar, and female adult. A relatively low dose (6 ng/insect) did not cause high mortality, retaining a high potential for gene silencing. Since RNAi is highly effective in this species, the deleterious effect of large amounts of dsRNA could be avoided by administering a low dsRNA dose.     

Gene silencing in mosquito salivary glands by RNAi

Salivary glands are the ultimate site of development in the insect of mosquito born pathogens such as Plasmodium. Mosquito salivary glands also secrete components involved in anti-haemostatic activities and allergic reactions. We investigated the feasibility of RNAi as a tool for functional analysis of genes expressed in Anopheles gambiae salivary glands. We show that specific gene silencing in salivary glands requires the use of large amounts of dsRNA, condition that differs from those for efficient RNAi in other mosquito tissues. Using this protocol, we demonstrated the role of AgApy, which encodes an apyrase, in the probing behaviour of An. gambiae.     

Comparison of dispersal ability between the green leafhoppers,Nephotettix virescens andN. cincticeps (hemiptera: Cicadellidae) by tethered flight

Dispersal propensity of green leafhoppers was compared between a tropical species, Nephotettix virescens, and a temperate species, N. cincticeps. The flight ability was measured with tethered flight technique under laboratory conditions (25°C, 16L-8D). The pre-flight period was shorter and the flight duration was longer in N. virescens than in N. cincticeps in both sexes. No significant correlations were found between the flight activity and morphometric characters for either of the two species. The results suggested that dispersal propensity of N. virescens is higher than that of N. cincticeps.     

Aberrations caused by neem oil odour in green leafhopper feeding on rice plants

Using an electronic device and a lignin-specific dye, the feeding behaviour of the green leafhopper,Nephotettix virescens (Distant), (Homoptera: Cicadellidae) was monitored on rice plants kept in an arena permeated with the odour of neem (Azadirachta indica A. Juss) seed oil. The garlicky odour of neem oil disrupted the normal feeding behaviour of the leafhopper. Phloem feeding byN. virescens on rice plants kept in an arena permeated with odour of 6, 12, or 25% neem oil was significantly reduced compared with that on control plants kept in an arena with paraffin oil or 1.66% aqueous Teepol solution. Reduced intake from phloem was associated with a significant increase in the insect's probing frequency and an increase in durations of salivation and xylem ingestion.

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Alterations du comportement sur riz de Nephotettix virescens, dues à l'odeur d'essence de neem (Azadirachta indica)

Résumé De nombreux essais en plein champ à l'I.R.R.I. ont révélé une réduction du T.V. (tungro virus) transmis par (Nephotettix virescens) sur des parcelles de riz sensible non-traitées, entourées de parcelles pulvérisées périodiquement avec de l'essence de graines de neem (Azadirachta indica).L'essence de neem a une forte odeur d'ail, perceptible à distance. Nous avons examiné l'effet de l'odeur d'essence de neem sur le comportement alimentaire deN. verescens, grâce à un dispositif électronique et un colorant spécifique de la lignine. Sur du riz maintenu dans une enceinte imprégnée d'odeur d'essence de neem, nous avons constaté une diminution de la consommation de phloème, une augmentation significative de la fréquence des piqûres et une augmentation de temps de salivation et d'absorption de xylème. Ces altérations du comportement alimentaire provoquées par l'odeur d'essence de neem peuvent expliquer la diminution de la fréquence du T.V. spécifique du phloème dans des champs de riz non-traités, entourés de champs pulvérisés avec de l'essence de neem.

     

Molecular mapping of a novel gene, Grh5, conferring resistance to green rice leafhopper (Nephotettix cincticeps Uhler) in rice, Oryza sativa L.

The green rice leafhopper (GRH), Nephotettix cincticeps Uhler, is one of the most serious insect pests affecting cultivated rice (Oryza sativa L.) in temperate regions of East Asia. An accession of the wild rice species, Oryza rufipogon Griff. (W1962), was found to be highly resistant to GRH by an antibiosis test. To understand the genetic basis of the GRH resistance, a BC₁F₁ population derived from a cross between a susceptible Japonica variety, Taichung 65 (T65), and a highly resistant accession W1962 was analyzed by quantitative trait loci (QTL) mapping. A single major QTL for GRH resistance was detected on rice chromosome 8. A nearly isogenic population containing segments of the targeted QTL region derived from W1962 was then developed through advanced backcrossing with marker-assisted selection. Further molecular mapping using a BC₄F₂ population revealed that a new resistance gene, designated as Green rice leafhopper resistance 5 (Grh5), was located on the distal region of the long arm of chromosome 8 and tightly linked to the simple sequence repeat markers RM3754 and RM3761. A nearly isogenic line (NIL) carrying Grh5 was subsequently developed in the progeny of the mapping population. The resistance level of Grh5-NIL was compared with those of developed NILs for GRH resistance and was found to have the highest resistance. The DNA markers found to be closely linked to Grh5 would be useful for marker-assisted selection for the improvement of resistance to GRH in rice.     

A tick B-cell inhibitory protein from salivary glands of the hard tick, Hyalomma asiaticum asiaticum

Some studies done to date suggest that B-cell inhibitory factor occurred in tick saliva. In this study, a novel protein having B-cell inhibitory activity was purified and characterized from the salivary glands of the hard tick, Hyalomma asiaticum asiaticum. This protein was named B-cell inhibitory factor (BIF). The cDNA encoding BIF was cloned by cDNA library screening. The predicted protein from the cDNA sequence is composed of 138 amino acids including the mature BIF. No similarity was found by Blast search. The lipopolysaccharide-induced B-cell proliferation was inhibited by BIF. This is the first report of the identification and characterization of B-cell inhibitory protein from tick. The current study facilitates the study of identifying the interaction among tick, Borrelia burgdorferi, the causative agent of Lyme disease, and host.