A computer simulation of the epidemiology of the rice dwarf virus

An epidemiological system model was developed to evaluate the role of factors which were responsible for the prevalence of rice dwarf virus (RDV) transmitted by the green rice leafhopper, Nephotettix cincticeps. Simulation tests were conducted by varying values of the following three parameters: the vector density, the coefficient of the efficiency of feeding acquisition of RDV of the vector, and that of efficiency of RDV transmission by the vector. The effect of each parameter was assessed in terms of changes in percentages of infected insects and of infected rice hills. Both the percentages of infected insects and of infected rice hills increased rapidly with increasing vector density within a range of low vector density. The former increased linearly when the acquisitive coefficient was increased. But the percentage of infected rice hills was affected to a lesser extent. The percentage of infected insects and that of infected rice hills increased exponentially with increasing values of the transmission coefficient. The results obtained from the simulation tests were discussed in relation to the ecological factors which caused the recent prevalence of RDV.     

Recovery of transgenic rice plants expressing the rice dwarf virus outer coat protein gene (S8)

 The coding region of the eighth largest segment (S8) of the rice dwarf virus (RDV) was obtained from a RDV Fujian isolate. It was then cloned into pTrcHisA for expression in E. coli and into vector pE3 for plant transformation. By using callus derived from mature rice embryos as the target tissue, we obtained regenerated rice plants after bombardment of the former with plasmid pE3R8 containing the RDV S8 gene and the marker gene neomycin phosphotransferase (NPT II). Southern blotting confirmed the integration of the RDV S8 gene into the rice genome. The expression of the outer coat protein in both E. coli and rice plants was confirmed by western blotting. The recovery of transgenic rice plants expressing S8 gene is an important step towards studying the function of the RDV genes and obtaining RDV-resistant rice plants. Received: 1 March 1996 / Accepted: 2 August 1996     

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.     

Bacteriome-associated endosymbionts of the green rice leafhopper <Emphasis Type="Italic">Nephotettix cincticeps</Emphasis> (Hemiptera: Cicadellidae)

The green rice leafhopper Nephotettix cincticeps (Uhler) is a commonly distributed pest of rice in East Asia. Early histological studies describe the presence of two bacteriome-associated symbionts and a rickettsial microorganism in N. cincticeps, but their microbiological affiliations have been elusive. We identified these bacterial symbionts using modern microbiological techniques. Cloning and sequencing of the 16S ribosomal RNA gene from dissected bacteriomes yielded two major and a minor bacterial sequences: a major sequence was placed in the Bacteroidetes clade of Sulcia muelleri, an ancient symbiont lineage associated with diverse hemipteran insects; another major sequence was allied to a β-proteobacterial sequence from a leafhopper Matsumuratettix hiroglyphicus; the minor sequence fell in the α-proteobacterial genus Rickettsia. In situ hybridization and transmission electron microscopy showed that the Sulcia symbiont and the β-proteobacterial symbiont are harbored within different types of bacteriocytes that constitute the outer and inner regions of the bacteriome, respectively. Oral administration of tetracycline to nymphal N. cincticeps resulted in retarded growth, high mortality rates, and failure in adult emergence, suggesting important biological roles of the symbionts for the host insect. The designation Candidatus Nasuia deltocephalinicola is proposed for the β-proteobacterial symbiont clade associated with N. cincticeps and allied leafhoppers of the subfamily Deltocephalinae.     

A comparative analysis on the distribution of nymphal populations of some leaf- and planthoppers on rice plant

The frequency distribution of the number of nymphs per hill of rice plant were analyzed for three species of rice leaf- and planthoppers, Nilaparvata lugens, Delphacodes striatella and Nephotettix cincticeps, based on the sampling data obtained during their last generations in the paddy field. For every species concerned, individual distributions were proved to be contagious and to fit well to the negative binomial distribution. Further, it was found that the value of negative binomial parameter k is so stable for same species that a single value of k is applicable for a series of counts with different means, whereas that k differs remarkably among different species: if the reciprocal of the weighted estimate of common k which is an adequate index measuring degree of contagiousness of the distribution, is compared among different species, it is higher in the order of Nilaparvata, Delphacodes and Nephotettix. The ecological and practical implication of constancy and heterogeneity within and among species was discussed respectively as to the value of parameter k.     

Spatio-temporal dynamics of <Emphasis Type="Italic">Scotinophara lurida</Emphasis> (Hemiptera: Pentatomidae) in rice fields

Studying the spatial pattern of insect pests and the temporal stability of their patterns is important in understanding underlying ecological mechanisms and in developing pest management programs in cultivated crop systems. To elucidate the spatio-temporal pattern of the black rice bug, Scotinophara lurida, in rice fields, samplings were conducted in two rice fields over 2 years. Using spatial analysis by distance indices, the spatial pattern of each developmental stage of S. lurida and their temporal stability of the spatial pattern were identified. Most of the I _a (the index of aggregation) values for overwintered adults and eggs of S. lurda were close to 1, indicating random distribution pattern while nymphs and new adults mainly had I _a values >1, indicating an aggregated distribution pattern. According to spatial association analysis between successive samples using X (the index of spatial association), the spatial pattern of S. lurida showed strong temporal stability throughout the season. Also, there was strong association between the spatial patterns of developmental stages, indicating the great effect of the spatial pattern of the previous developmental stage on that of later developmental stage. Factors influencing the spatial pattern and spatial stability of S. lurida are discussed.     

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.     

The role of amphibian prey in the diet and growth of giant water bug nymphs in Japanese rice fields

Predatory insects that depend upon particular prey animals are commonly regulated by the prey animal’s abundance. Nymphs of the giant water bug Kirkaldyia (=Lethocerus) deyrolli (Heteroptera: Belostomatidae) are predators regarded as specialists in feeding on tadpoles. We studied the ontogenetic diet shift of aquatic nymphs by quantifying instar abundance and by analyzing captured prey and prey relative abundance during the period of rice irrigation in three localities. We also evaluated the contribution of major prey items (tadpoles, frogs, and Odonata nymphs) on specific growth rates of each nymphal stage in a rearing experiment. First to third-instar nymphs of K. deyrolli fed mainly on tadpoles, regardless of differences in prey availability. Nymphs of subsequent fourth and fifth instar stages shifted from tadpoles to other prey animals within each rice field. A rearing experiment demonstrated that giant water bug nymphs provided with tadpoles had greater specific growth rates at all nymphal stages, except for the final stage, than nymphs fed other prey (frogs and Odonata nymphs). The emergence of young K. deyrolli nymphs seemed to coincide with the period during which tadpoles became abundant in the rice fields. Consumption of tadpoles seems important to allow the nymph to complete its larval development in an unstable temporary habitat. An erratum to this article can be found at     

Effect of mating combinations on the host parasitisation and sex allocation in solitary endoparasitoid,Aenasius arizonensis (Hymenoptera: Encyrtidae)

Aenasius arizonensis (Girault) is an important solitary endoparasitoid of Phenacoccus solenopsis Tinsley. To optimise the mass production of high-quality females, it is important to assess the influence of mating regimes on the progeny fitness and sex allocation. We, therefore, hypothesise that mating combinations in A. arizonensis adults emerged from different host instars may influence parasitism and sex allocation in the subsequent generation. Therefore, we compared three nymphal instars (1st, 2nd and 3rd) and adults host stages of P. solenopsis for parasitism and sex allocation by A. arizonensis. Further, F₁ female progeny of the parasitoid emerged from different host instars was henceforth evaluated for its fitness in six mating combinations. A. arizonensis females parasitised all the host stages except the 1st instar nymphs. The parasitised 2nd instar nymphs yielded only males, while the sex ratio in the later host instars was strongly female-biased. The parasitoid females preferred 3rd instar nymphs with respect to higher parasitism (74.0–84.0%) and produced more females in the F₁ progeny as compared to other host stages. F₁ females that emerged from 3rd instar nymphs produced significantly higher parasitism (74.0–79.0%). These mating combinations also yielded more female progeny in the F₂ generation. However, parasitism by F₁ females was significantly lower (9.0–12.0%) when mated with males that emerged from 2nd instar P. solenopsis nymphs. Moreover, latter combinations yielded only male progeny in F₂ generation. These findings can be used in laboratory mass rearing of this parasitoid vis-à-vis biological control of P. solenopsis.     

Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector

Background

Rice dwarf virus (RDV), a plant reovirus, is mainly transmitted by the green rice leafhopper, Nephotettix cincticeps, in a persistent-propagative manner. Plant reoviruses are thought to replicate and assemble within cytoplasmic structures called viroplasms. Nonstructural protein Pns4 of RDV, a phosphoprotein, is localized around the viroplasm matrix and forms minitubules in insect vector cells. However, the functional role of Pns4 minitubules during viral infection in insect vector is still unknown yet.

Methods

RNA interference (RNAi) system targeting Pns4 gene of RDV was conducted. Double-stranded RNA (dsRNA) specific for Pns4 gene was synthesized in vitro, and introduced into cultured leafhopper cells by transfection or into insect body by microinjection. The effects of the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene on viral replication and spread in cultured cells and insect vector were analyzed using immunofluorescence, western blotting or RT-PCR assays.

Results

In cultured leafhopper cells, the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene strongly inhibited the formation of minitubules, preventing the accumulation of viroplasms and efficient viral infection in insect vector cells. RNAi induced by microinjection of dsRNA from Pns4 gene significantly reduced the viruliferous rate of N. cincticeps. Furthermore, it also strongly inhibited the formation of minitubules and viroplasms, preventing efficient viral spread from the initially infected site in the filter chamber of intact insect vector.

Conclusions

Pns4 of RDV is essential for viral infection and replication in insect vector. It may directly participate in the functional role of viroplasm for viral replication and assembly of progeny virions during viral infection in leafhopper vector.

     

Life table studies on the paddy field population of the green rice leafhopper,Nephotettix cincticeps Uhler (Hemiptera: Cicadellidae), with special reference to the mechanism of population regulation

The population growth of the green rice leafhopper, Nephotettix cincticeps, in the paddy field was analyzed based on the life table data accumulated for six years. The paddy field population, which stems from the invading adults of the first generation (G-I), repeats two complete generations, and the hatchlings of the fourth generation (G-IV) enter diapause and overwinter as the fourth instar nymphs in fallow paddy fields. It was clarified that the density dependent reduction in the mean longevity and oviposition rate of adult females in G-II and III played a primary role in stabilizing the annual population densities. The annual average of the mean longevity of G-II females (3.9 days) was much shorter than that of G-III ones (7.7 days) and thus the density dependent reduction in the mean longevity induced a more prompt regulatory effect on the oviposition of G-II females compared with G-III ones. As the result, two equilibrium densities of eggs were obtained, e.g., ca 100 and 700 eggs per hill in G-III and IV, respectively. Density dependent decrease in the proportion of mature females in the adult population was especially conspicuous in G-II, and this was closely associated with the density dependent reduction in the mean longevity and fecundity. Thus, the density dependent dispersal (emigration) of the adult females by flight in G-II and III was the most convincing factor in the process of population regulation. The density dependent dispersal of the adult females is effective in avoiding the deleterious effects of nymphal crowding in a breeding habitat unit (a paddy field), and may result in a more even distribution of the population over a continuous habitat units in a locality than otherwise.     

Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests,Nilaparvata lugens andNephotettix cinciteps

Insect feeding trials were carried out to determine the effects of incorporating a range of plant derived proteins into artificial diets fed to leafhopper and planthopper pests of rice. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soy bean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål) when incorporated into artificial diet at 0.1% (w/v), 0.1% (w/v) and 0.08% (w/v) levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephotettix cinciteps Uhler). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects.     

Patterns of phytoplasma-infected and infective Scaphoideus titanus leafhoppers in vineyards with high incidence of Flavescence dorée

The incidence and transmissibility of Flavescence dorée phytoplasma (FDP) in populations of the vector Scaphoideus titanus Ball (Homoptera: Cicadellidae) were investigated by periodically collecting nymphs and adults of the leafhopper species in four vineyards with high incidence of Flavescence dorée (FD)‐diseased grapevines. Insects were tested individually for FDP with an ELISA procedure, after transmission assays to broadbean seedlings and further transmission to grapevine cuttings. No transmission occurred when early or middle instar nymphs were used to inoculate broadbeans, although a limited number of fifth‐instar nymphs and young adults transmitted the pathogen to broadbean seedlings. However, the same batches of insects transmitted FDP more efficiently to grapevine cuttings during prolonged inoculation periods, confirming the existence of a latent period before infected insects become infective. The proportions of ELISA‐positive individuals in the three categories of insects used for transmission assays reflected the rate of FDP transmission to grapevine cuttings. According to the data obtained by ELISA and from field sampling of first‐instar nymphs, we adapted the proportions of nymph hatching, of infected leafhoppers, and of infective leafhoppers (assuming a conservative latent period in the vector of 30 days) to logistic models as a function of degree‐days. We then discussed the possible use of the model developed for improving vector control decisions in FD‐infected vineyards.     

Bt rice expressing Cry1Ab does not stimulate an outbreak of its non-target herbivore, Nilaparvata lugens

In this study, the non-target effects of Bt rice “KMD2” expressing a Cry1Ab protein on the performance of the brown planthopper (BPH), Nilaparvata lugens, over multiple generations were evaluated under laboratory and field conditions. In the laboratory, BPH was reared to observe the impact of the Bt rice as compared to its parental non-Bt cultivar Xiushui 11, while the population dynamics and oviposition performance of BPH were investigated in the field. The survival of BPH nymphs fed Bt and non-Bt rice did not differ significantly. The nymph developmental duration of BPH was significantly delayed by the Bt rice by comparison with the non-Bt rice for the 1st and 2nd but not the 4th generation. Most importantly, the fecundity of BPH on the Bt rice was significantly decreased in every generation when compared with the non-Bt rice. In the field investigations, the population density of BPH nymphs was significantly lower in the Bt rice field. However, the temporal pattern of population dynamics of BPH adults was similar between the Bt and non-Bt rice, presumably due to migratory interference of the adults. In the Bt rice field, the percentage of tillers with eggs and the number of eggs per tiller were also significantly lower from tillering to mature stage. Additionally, Cry1Ab protein could not be detected in guts from single BPH adults. In general, our results suggest that the Bt rice “KMD2” could not stimulate an outbreak of BPH.     

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.     

Genetics of gel consistency in rice (Oryza sativa L.)

Inheritance of gel consistency in rice was studied in crossés involving highamylose, low-gelatinizalion temperature parents with hard, medium, and soft gel consistency. The results of single-grain analysis of parents, F₁, F₂, B₁F₁, B₂F₂, and their reciprocal crosses from a single-season harvest showed that the differences between hard and soft, hard and medium, and medium and soft gel consistency are under monogenic control and that modifiers affect the expression of the trait. Multiple alleles at the same locus, hereby designated asgec ^a for medium gel consistency andgec ^b for soft gel consistency, were recessive to the wild type allele for hard gel consistency andgec ^a was dominant overgec ^b. The results indicate that selection for desired gel consistency can effectively be done in early segregating generations.     

The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo, leading to reduced biosynthesis of gibberellins and rice dwarf symptoms

The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development.     

Effect of talc-formulated entomopathogenic fungus Beauveria against leaffolder (Cnaphalocrosis medinalis) in rice

Thirteen Beauveria strains were isolated from the soil and infected insects. Among the various isolates, B₂ isolate (Arachalore) showed a higher percentage of mortality against C. medinalis (73.3%) under in vitro conditions. Conidial concentration of 1 × 10⁸ of the B2 strain registered maximum mortality of 76.7%. The least LT₅₀ value of 4.4 days was registered in B2 isolate with the spore concentration of 1 × 10⁸ and the LC₅₀ value was 3.4 × 10⁴. Beauveria strains altered the feeding behavior of C. medinalis, reduced the pupal weight, prolonged the pupation period, malformed the pupa and adult under in vitro. The efficacy of the talc-based bioformulation of Beauveria (B2) strain was tested as seed treatment + seedling dip + soil application + foliar spray against rice leaffolder under in vitro and greenhouse conditions. The percentage damage was significantly less (5.5) in B2 as compared to untreated healthy control (25.8). In addition, the same treatment increased the activities of defense-related enzymes, namely peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, chitinase, and phenolics in rice.     

Artificial rearing of the rice green leafhopper, Nephotettix cincticeps, on a holidic diet

The rice green leafhopper, Nephotettix cincticeps, was reared from the first instar to the adult stage on a holidic diet. Adult emergence from the diet-reared nymphs was ca. 53.3%, similar to rice seedlingreared; nymphal period being 22.5 days at 25°. The diet-reared females did not lay eggs under artificial feeding conditions although they had normal eggs in their ovarioles and could oviposit on rice seedlings in a test tube. The diet containing 1.68% (w/v) amino acid mixture and 5% (w/v) sucrose was best for growth and development.

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Résumé Nephotettix cincticeps a été élevé des larves venant d'éclore à l'adulte sur un régime holidique. Le taux d'adulte obtenus (53,3%) est voisin de celui obtenu avec élevage sur pousses de riz. Le développement larvaire demande 22,5±0,4 jours à 25°. Les femelles élevées avec ce régime ne pondent pas d'ufs en alimentation artificielle bien qu'elles aient un nombre normal d'ufs dans les ovarioles et pondent sur pousses de riz dans un tube témoin. Un régime contenant 1,68% d'acides aminés mélangés et 5% de sucrose assure la meilleure croissance et le développement le plus rapide.