Transovarial Transmission of a Plant Virus Is Mediated by Vitellogenin of Its Insect Vector

Most plant viruses are transmitted by hemipteroid insects. Some viruses can be transmitted from female parent to offspring usually through eggs, but the mechanism of this transovarial transmission remains unclear. Rice stripe virus (RSV), a Tenuivirus, transmitted mainly by the small brown planthopper (Laodelphax striatellus), is also spread to the offspring through the eggs. Here, we used the RSV–planthopper system as a model to investigate the mechanism of transovarial transmission and demonstrated the central role of vitellogenin (Vg) of L. striatellus in the process of virus transmission into the eggs. Our data showed Vg can bind to pc3 in vivo and in vitro and colocalize in the germarium. RSV filamentous ribonucleoprotein particles (RNPs) only accumulated in the terminal filaments and pedicel areas prior to Vg expression and was not present in the germarium until Vg was expressed, where RSV RNPs and Vg had colocalized. Observations by immunoelectron microscopy (IEM) also indicated that these two proteins colocalized in nurse cells. Knockdown of Vg expression due to RNA interference resulted in inhibition of the invasion of ovarioles by RSV. Together, the data obtained indicated that RSV RNPs may enter the nurse cell of the germarium via endocytosis through binding with Vg. Finally, the virus enters the oocytes through nutritive cords, using the same route as for Vg transport. Our results show that the Vg of L. striatellus played a critical role in transovarial transmission of RSV and shows how viruses can use existing transovarial transportation systems in insect vectors for their own purposes.     

Characterization of Rice Black-Streaked Dwarf Virus- and Rice Stripe Virus-Derived siRNAs in Singly and Doubly Infected Insect Vector Laodelphax striatellus

Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi) which generates viral-derived small interfering RNAs (siRNAs). However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus) was infected by Rice black-streaked dwarf virus (RBSDV) (Reoviridae; Fijivirus), more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV), a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5′- and 3′-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.     

Coordination between terminal variation of the viral genome and insect microRNAs regulates rice stripe virus replication in insect vectors

Maintenance of a balance between the levels of viral replication and selective pressure from the immune systems of insect vectors is one of the prerequisites for efficient transmission of insect-borne propagative phytoviruses. The mechanism regulating the adaptation of RNA viruses to insect vectors by genomic variation remains unknown. Our previous study demonstrated an extension of the 3’-untranslated terminal region (UTR) of two genomic segments of rice stripe virus (RSV). In the present study, a reverse genetic system for RSV in human cells and an insect vector, the small brown planthopper Laodelphax striatellus, was used to demonstrate that the 3’-terminal extensions suppressed viral replication in vector insects by inhibiting promoter activity due to structural interference with the panhandle structure formed by viral 3’- and 5’-UTRs. The extension sequence in the viral RNA1 segment was targeted by an endogenous insect microRNA, miR-263a, which decreased the inhibitory effect of the extension sequence on viral promoter activity. Surprisingly, the expression of miR-263a was negatively regulated by RSV infection. This elaborate coordination between terminal variation of the viral genome and endogenous insect microRNAs controls RSV replication in planthopper, thus reflecting a distinct strategy of adaptation of phytoviruses to insect vectors.     

Silence of the killers: discovery of male-killing suppression in a rearing strain of the small brown planthopper,Laodelphax striatellus

According to evolutionary theory, sex ratio distortions caused by reproductive parasites such as Wolbachia and Spiroplasma are predicted to be rapidly normalized by the emergence of host nuclear suppressors. However, such processes in the evolutionary arms race are difficult to observe because sex ratio biases will be promptly hidden and become superficially unrecognizable. The evolution of genetic suppressors has been reported in just two insect species so far. In the small brown planthopper, Laodelphax striatellus, female-biases caused by Spiroplasma, which is a ‘late’ male-killer, have been found in some populations. During the continuous rearing of L. striatellus, we noted that a rearing strain had a 1 : 1 sex ratio even though it harboured Spiroplasma. Through introgression crossing experiments with a strain lacking suppressors, we revealed that the L. striatellus strain had the zygotic male-killing suppressor acting as a dominant trait. The male-killing phenotype was hidden by the suppressor even though Spiroplasma retained its male-killing ability. This is the first study to demonstrate the existence of a late male-killing suppressor and its mode of inheritance. Our results, together with those of previous studies, suggest that the inheritance modes of male-killing suppressors are similar regardless of insect order or early or late male killing.     

Seasonal occurrence of Laodelphax striatellus (Hemiptera: Delphacidae) in a rice-forage crops mixed cropping area in central Kyushu,Japan

The small brown planthopper, Laodelphax striatellus (Fallén) is an important pest of rice in East Asia because it is a vector of rice stripe virus. In this study, we examined the seasonal occurrence of L. striatellus in an agricultural area in central Kyushu in which cropping systems have been changing since the 1970s. The first generation of L. striatellus emerged in the spring, not only on barley, oats, and Italian ryegrass but also on spring crops of forage maize. The second generation of L. striatellus adults emerged in forage maize fields in July, after harvesting of winter crops of barley, oats, and Italian ryegrass. These results suggest that spring crops of forage maize are important hosts of L. striatellus from spring to early summer. The overwintering generation of L. striatellus was collected from rice ratoon, suggesting this is a favorable host for L. striatellus during winter. The appearance of these novel hosts may be a factor in the recent re-emergence of L. striatellus in central Kyushu.     

Prediction of overseas migration of the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae) in East Asia

A method has been developed for predicting the overseas migration of Laodelphax striatellus (Fallén) from eastern China to Japan and Korea. The method consists of two techniques: estimation of the emigration period in the source region and simulation of migration. The emigration period was estimated by calculating the effective accumulated temperature for the insect by use of real-time daily surface temperatures at the source. During the emigration period, migration simulations were performed twice a day, at every dusk and dawn. The prediction method was evaluated, by cross-validation using migrations in the 4 years from 2008 to 2011. The results showed that the emigration periods included the mass migrations, and that the method successfully predicted those migrations.     

Efficient monitoring of maize orange leafhopper, <Emphasis Type="Italic">Cicadulina bipunctata</Emphasis> (Hemiptera: Cicadellidae), and small brown planthopper, <Emphasis Type="Italic">Laodelphax striatellus</Emphasis> (Hemiptera: Delphacidae), in forage maize fields using yellow sticky traps

The monitoring of insect pests in fields of forage maize is difficult because plants are tall and grow at a high density. We investigated the effectiveness of colored sticky traps and appropriate conditions for monitoring insect pests in forage maize fields. Large numbers of the maize orange leafhopper, Cicadulina bipunctata Melichar (Hemiptera: Cicadellidae), and the small brown planthopper, Laodelphax striatellus Fallen (Hemiptera: Delphacidae), were collected during the experimental period with yellow and blue sticky traps placed in summer crop forage maize fields. A greater number of insects were trapped in yellow traps relative to blue traps. Traps located at a lower height (40 cm above the ground) attracted larger numbers of C. bipunctata, whereas L. striatellus did not demonstrate a height-dependent preference. These results indicated that yellow-colored sticky traps located at low height are effective for collecting C. bipunctata and L. striatellus simultaneously. Seasonal occurrence data obtained by the yellow sticky traps showed clearer seasonal occurrences than that obtained by two previously developed methods, suction and light traps, indicating that sticky traps are effective for monitoring the seasonal occurrence of these two insects in forage maize fields.     

Sterols in Laodelphax striatellus with special reference to the intracellular yeastlike symbiotes as a sterol source

Sterols were analysed to investigate the sterol source in Laodelphax striatellus and three other rice plant-sucking homopterous insects. In L. striatellus, cholesterol, 24-methylenecholesterol and β-sitosterol were detected. The host plant (rice) contained campesterol, stigmasterol and β-sitosterol. From the honeydew excreted by L. striatellus, cholesterol, β-sitosterol and negligible amounts of campesterol were recovered. Laodelphax striatellus possesses yeastlike symbiotes which can be destroyed by high temperature. Fifth instar nymphs, which have been exposed to 35°C for 3 days in their 1 st instar, showed lower cholesterol concentration and markedly reduced amounts of 24-methylenecholesterol. From the results it is concluded that L. striatellus has two sterol sources: one from the host plant and the other from the yeastlike symbiotes which appear to provide 24-methylenecholesterol.     

基于rDNA ITS1和ITS2序列的褐飞虱、白背飞虱和灰飞虱的分子鉴定

本研究测定了褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera和灰飞虱Laodelphax striatellus的rDNA ITS1和ITS2的序列, 以探讨这3种稻飞虱的分子鉴定方法。3种飞虱的ITS1和ITS2侧翼区（18S, 5.8S和28S）序列相对稳定, 但ITS1和ITS2序列在3种飞虱中变异较大。 ITS1在所分析的438个位点中可变位点达294个, ITS2在分析的403个位点中可变位点为177个。根据3种飞虱rDNA的ITS1和ITS2序列设计了特异性引物, 应用特异性引物对样品进行了PCR扩增, 分析发现3种飞虱ITS1区的特异性引物扩增效果不理想, 而ITS2区的特异性引物可以稳定地扩增出明显的目的DNA条带. 因此, 采用ITS2区的特异性引物可以对3种飞虱进行快速的分子鉴定。     

Diapause Induction and Termination in the Small Brown Planthopper,Laodelphax striatellus (Hemiptera: Delphacidae)

The small brown planthopper, Laodelphax striatellus (Fallén) enters the photoperiodic induction of diapause as 3rd or 4th instar nymphs. The photoperiodic response curves in this planthopper showed a typical long-day response type with a critical daylength of approximately 11 h at 25°C, 12 h at 22 and 20°C and 12.5 h at 18°C, and diapause induction was almost abrogated at 28°C. The third stage was the most sensitive stage to photoperiod. The photoperiodic response curve at 20°C showed a gradual decline in diapause incidence in ultra-long nights, and continuous darkness resulted in 100% development. The required number of days for a 50% response was distinctly different between the short- and long-night cycles, showing that the effect of one short night was equivalent to the effect of three long nights at 18°C. The rearing day length of 12 h evoked a weaker intensity of diapause than did 10 and 11 h. The duration of diapause was significantly longer under the short daylength of 11 h than it was under the long daylength of 15 h. The optimal temperature for diapause termination was 26 and 28°C. Chilling at 5°C for different times did not shorten the duration of diapause but significantly lengthened it when chilling period was included. In autumn, 50% of the nymphs that hatched from late September to mid-October entered diapause in response to temperatures below 20°C. The critical daylength in the field was between 12 h 10 min and 12 h 32 min (including twilight), which was nearly identical to the critical daylength of 12.5 h at 18°C. In spring, overwintering nymphs began to emerge in early March-late March when the mean daily temperature rose to 10°C or higher.     

Overexpression of Rice Black-Streaked Dwarf Virus P7-1 in Arabidopsis Results in Male Sterility Due to Non-Dehiscent Anthers

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, is propagatively transmitted by the small brown planthopper (Laodelphax striatellus Fallén). RBSDV causes rice black-streaked dwarf and maize rough dwarf diseases, which lead to severe yield losses in crops in China. Although several RBSDV proteins have been studied in detail, the functions of the nonstructural protein P7-1 are still largely unknown. To investigate the role of the P7-1 protein in virus pathogenicity, transgenic Arabidopsis thaliana plants were generated in which the P7-1 gene was expressed under the control of the 35S promoter. The RBSDV P7-1-transgenic Arabidopsis plants (named P7-1-OE) were male sterility. Flowers and pollen from P7-1-transgenic plants were of normal size and shape, and anthers developed to the normal size but failed to dehisce. The non-dehiscent anthers observed in P7-1-OE were attributed to decreased lignin content in the anthers. Furthermore, the reactive oxygen species levels were quite low in the transgenic plants compared with the wild type. These results indicate that ectopic expression of the RBSDV P7-1 protein in A. thaliana causes male sterility, possibly through the disruption of the lignin biosynthesis and H₂O₂-dependent polymerization pathways.     

Current status of insecticide resistance in the small brown planthopper, <Emphasis Type="Italic">Laodelphax striatellus</Emphasis>, in Japan,Taiwan, and Vietnam

The small brown planthopper, Laodelphax striatellus, is one of the most serious pest insects of rice plants. A large migration of the insects from overseas was reported in western parts of Japan in June 2008. Insecticide resistance to imidacloprid, fipronil and BPMC was compared among local populations in these western regions after migration. The insecticides were applied to the insects using a topical application method. In some populations, the resistance status coincided with that of the immigrant insects just after migration, i.e., resistance to imidacloprid but susceptibility to fipronil. In other populations, resistance was observed not only against imidacloprid but also fipronil. It is likely that the status of the latter populations resulted from intercrossing between domestic populations of the insects and migrants. Insecticide resistance was also assessed in other areas of northern and eastern parts of Japan. In general, these populations showed relatively low resistance, although resistance to fipronil was high in the eastern part of Japan where the density of domestic populations has recently increased. Insecticide susceptibilities were also assessed in several sites in Taiwan and the northern parts of Vietnam. Although susceptibilities differed among these sites or countries, they have recently seen a decline for all three insecticides.     

Development and preliminary application of a triplex real‐time polymerase chain reaction assay for evaluating predation on three planthoppers in a rice ecosystem

A multiplex real‐time quantitative polymerase chain reaction (PCR) assay was developed to simultaneously detect the DNA of three rice planthoppers, that is, Sogatella furcifera (Horváth) (white‐backed planthopper), Nilaparvata lugens (Stål) (brown planthopper) and Laodelphax striatellus (Fallén) (small brown planthopper), in the gut of their predators. The sets of primers and ALLGlo probes were targeted to the regions of internal transcribed spacer 2 (ITS2) genes in nuclear ribosomal DNA (rDNA). The sensitivity, specificity and interference test for the multiplex real‐time quantitative PCR assay were analysed. The assay's detection limits were 100, 1000 and 100 copies for the white‐backed planthopper, the brown planthopper and the small brown planthopper, respectively. The specificity tests showed no cross‐reactivity with genomic DNA from 30 other dominant herbivores, saprophagous insects and predators from rice ecosystem for each planthopper species. The assay was used in a preliminary study of predation events on the three planthoppers by three major spiders viz., Pardosa pseudoannulata (Bösenberg et Strand), Ummeliata insecticeps (Bösenberg et Strand) and Tetragnatha maxillosa Thorell which each differ in their preferred microhabitat as well as their predatory habits in rice field, and the results showed their predation on each planthopper species could be well evaluated using this method. Therefore, the multiplex real‐time quantitative PCR assay provides a new tool to study the mechanisms of prey shifting and natural regulation of the three rice planthoppers by generalist predators in rice ecosystem.     

Maize Iranian mosaic virus infection promotes the energy sources of its insect vector,Laodelphax striatellus

Maize Iranian mosaic virus (MIMV, family Rhabdoviridae) causes an important disease in cereal crops in Iran. It is transmitted by the planthopper Laodelphax striatellus in a persistent, propagative manner. In the present study, the effect of MIMV on the energy reserves of L. striatellus was studied by comparing energy contents in viruliferous and non‐viruliferous insects. Results showed that MIMV‐infected male and female adults, and nymphs stored 1.82, 2.24 and 1.7‐fold more total energy reserves than non‐viruliferous individuals. This is consistent with a 2.55‐fold increase of the total energy (sum of energy sources in nymphs and adults) of viruliferous compared to non‐viruliferous specimens. A significant increase in glycogen (2.26‐fold), carbohydrate (2.13‐fold), lipid (1.63‐fold) and protein (1.96‐fold) was documented in viruliferous insects compared to non‐viruliferous insects. Based on these results, we conclude that MIMV enhances the energy reserves of its vector and therefore, may play a vital role in the ecology of L. striatellus.     

The long-distance migration of Nilaparvata lugens (Stål) (Delphacidae) in China: radar observations of mass return flight in the autumn

Abstract.

• 1 A case study is presented of the autumn migration of the brown planthopper, Nilaparvata lugens (Stål), in the area of Nanjing in the People's Republic of China. The study was made using a high frequency (8 mm wavelength) radar and a net suspended from a kytoon.
• 2 The observations confirmed that long-distance return migrations occur in China in mid and late September, with N.lugens being carried on the prevailing north-easterly wind towards the autumn infestation and overwintering areas of the species.
• 3 After mass take-off in the late afternoon or at dusk, the migrants flew for several hours during the evening, often in a dense layer which formed at heights between about 400 and 1000m above ground. These layers often had well-defined ceilings corresponding to an air temperature of about 16°C. The migration height was above the top of the surface temperature inversion, i.e. the migrants did not fly at the height of the warmest air.
• 4 The dense layer concentrations overflying the radar were backtracked to source areas up to 240 km away in the north-east of Jiangsu Province. Planthoppers observed emigrating from the Nanjing area would reach areas in south Anhui Province or north Jiangxi Province if they flew for 12 h.
• 5 There was a second period of mass take-off at dawn. Insect layers sometimes formed but did not last longer than 1–2h.
• 6 The present results were strikingly different from those previously observed in the dry season in the Philippines, where migratory flight durations were largely confined to periods of about 30min at dusk and dawn.
• 7 Our observations are discussed in relation to the equator-wards return migrations undertaken in autumn by other insect species, and the importance of these migrations for the maintenance of long-flying genotypes in the overwintering populations is considered.

     

Knockdown of a putative Halloween gene Shade reveals its role in ecdysteroidogenesis in the small brown planthopper Laodelphax striatellus

Ecdysteroid hormone 20-hydroxyecdysone (20E) plays fundamental roles in insect development and reproduction, whereas the primary role of ecdysone (E) is the precursor for 20E. A cytochrome P450 monooxygenase (CYP), encoded by a Halloween gene Shade (Shd, cyp314a1), catalyzes the conversion of E into 20E in representative insect species in Diptera, Lepidoptera and Orthoptera. We describe here the cloning and characterization of LsShd in a hemipteran insect species, the small brown planthopper Laodelphax striatellus. LsSHD has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal expression pattern of LsShd was determined through the fourth-instar and the early fifth-instar stages by qPCR. LsShd showed two expression peaks in day 2 and day 5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Dietary introduction of double-stranded RNA (dsRNA) of LsShd into nymphs successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, and caused nymphal lethality and delayed development. Ingestion of 20E did not increase LsShd expression level, but almost completely rescued LsEcR mRNA level, and relieved the negative effects on the survival and development in LsShd-dsRNA-exposed nymphs. In contrast, dietary introduction of E had little rescue effects. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects, and LsSHD functions to regulate metamorphotic processes by converting E to 20E even in a hemipteran insect, L. striatellus.     

Cytoplasmic incompatibility in allopatric field populations of the small brown planthopper, Laodelphax striatellus, in Japan

The small brown planthopper, Laodelphax striatellus (Homoptera, Delphacidae), shows reproductive cytoplasmic incompatibility between laboratory-cultured strains. Wild planthoppers, were crossed for the purpose of examining the incompatibility principle in allopatric field populations. On the basis of the incompatibility recorded in field populations, Japanese planthopper populations were divided into two groups: one from the north-eastern part and one from the western part of Japan. Crosses between the females of the former group and males of the latter gave infertile eggs, showing unidirectional incompatibility. A small number of eggs from incompatible pairs, however, developed normally.

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Résumé Laodelphax striatellus présente une incompatibilité cytoplasmique reproductive entre souches de laboratoire. Des individus de la nature ont été croisés pour examiner l'incompatibilité entre populations allopatriques récoltées dans les champs. Sur la base de l'incompatibilité observée avec les populations naturelles, les populations japonaises ont été divisées en deux groupes: un pour la partie nordest du Japon, l'autre pour la partie occidentale. Les croisements entre femelles du premier groupe et mâles du second ont donné des oeufs stériles, montrant une incompatibilité unilatérale. Un petit nombre d'oeufs de couples incompatibles se sont cependant développés normalement.