High Temperature Effects on Yeast-like Endosymbiotes and Pesticide Resistance of the Small Brown Planthopper, Laodelphax striatellus

The newly-hatched nymphs of the small brown planthopper (SBPH), Laodelphax striatellus, including field and sensitive populations, were subjected to the high-temperature (35°C) treatment. The number of yeast-like endosymbiotes in SBPH reduced by 23.47%–34.23%, 57.86%–61.51% and 88.96%–90.71% after the high-temperature treatment for 1 d, 2 d, and 3 d, respectively. However, the size of yeast-like endosymbiotes was not obviously affected. Resistance of SBPH to three insecticides (imidacloprid, chlorpyrifos and fipronil) decreased with the increase of treatment time.

Identification and characterization of a TLR13 gene homologue from Laodelphax striatellus involved in the immune response induced by rice stripe virus

Toll-like receptors(TLRs) are the critical superfamily homologues that initiate sensing of the invasion of pathogens by the Toll pathway. As one of several intracellular nucleic acid-sensing TLRs, TLR13 is activated by an unmethylated motif present in the large ribosomal subunit of bacterial RNA. However, little attention has been paid to the function of TLR13 gene homologue from Laodelphax striatellus(designated as LsToll-13) in the immune response to rice stripe virus(RSV). Herein, LsToll-13 was cloned and characterized using RACE-PCR. Phylogenetic analysis showed that LsToll-13 was clustered with the TLR13 from six insects. Real-time PCR analysis demonstrated that the expression level of LsToll-13 was significantly reduced in L. striatellus with RSV infection compared with that in the naive strain. When the expression of LsToll-13 was significantly up-regulated at 6 h after bacterial infection, the expression of ribonucleoprotein(RNP) indicated that the RSV titer in the host insect was significantly suppressed. Upon knockdown of LsToll-13, using RNA interference(RNAi) in L. striatellus, the expression level of RNP was significantly increased with enhanced RSV accumulation, suggesting that LsToll-13 potentially protects L. striatellus from RSV infection. Taken together, our results indicated that LsToll-13 might be involved in the immune response of L. striatellus to RSV infection, and provided a new insight into further elucidating the molecular mechanisms of complex pathogen-host interactions and integrative pest management.

Subcellular localization of the stripe disease-specific protein encoded by rice stripe virus (RSV) in its vector,the small brown planthopper,Laodel-phax striatellus

The stripe disease-specific protein (SP) encoded by the rice stripe virus (RSV) was successfully used as a localization signal of the virus in its vector, the small brown lanthopper, Laodelphax striatellus Fallen. Immunogold particles in large numbers were detected in various parts of the viruliferous females: the ovum, surface of chorion, the mid-gut lumen, and the columnar cells. Whereas there was noneof these particles in the non-viruliferous females and males, and testis of viruliferous males. Endosymbionts (mycetocytes) were abundant, harboring ovaries of both viruliferous and non-viruliferous females, but none in the testis of males. The results provided us with the direct proof that RSV is a ciruculative and propagative plant virus and it was transovarially transmitted alongside with endosymbionts of its vector. Therefore, we deem it a nice lead for future studies on the mechanism of RSV transmission and functioning of its viral proteins.

Antibiotic exposure perturbs the bacterial community in the small brown planthopper Laodelphax striatellus

Bacteria symbionts in herbivores play an important role in host biology and ecology,and are affected by environmental factors such as temperature,diet,habitat,antibiotics and so on.However,the effects of antibiotics on the microbiome of the small brown planthopper Laodelphax striatellus(SBPH)remain unclear.Here,we studied the effects of tetracycline on the diversity and composition of bacterial colonies in different tissues of SBPH using high throughput sequencing of 16S ribosomal RNA amplicons.Our results show that Proteobacteria,Firmicutes,Bacteroidetes,Actinobacteria were most abundant in SBPH,and the genera Asaia and Wolbachia were most abundant in all body parts of SBPH.Antibiotic treatment had persistent effects on the composition of the SBPH microbiome.Tetracycline depleted the population of Firmicutes,Bacteroidetes,Tenericutes and Fusobacteria,and nearly 100%eliminated Wolbachia,Bacteroides and Abiotrophia in SBPH.Together,these results suggest that antibiotic exposure affects the bacteria symbionts of different body parts in SBPH and will facilitate future studies of the bacterial symbionts of arthropod hosts.

Molecular cloning and characterization of the putative Halloween gene Phantom from the small brown planthopper Laodelphax striatellus

Ecdysteroid hormone 20-hydroxyecdysone plays fundamental roles in in- sect postembryonic development and reproduction. Several cytochrome P450 mono- oxygenases （CYPs）, encoded by the Halloween genes, have been documented to be involved in ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. Here the putative Halloween gene Phantom （Phm, cyp306al） from a hemipteran insect species, the small brown planthopper Laodelphax striatellus, was cloned. LsPHM shows five insect conserved P450 motifs, that is, Helix-C, Helix-I, Helix-K, PERF and heme- binding motifs. Temporal and spatial expression patterns of LsPhm were evaluated by quantitative polymerase chain reaction. Through the fourth-instar and the early fifth-instar stages, LsPhm showed two expression peaks in day 2 and days 4-5 fourth-instar nymphs, and three troughs in day 1 and 3 fourth instars and day 1 fifth instars. On day 5 of the fourth-instar nymphs, LsPhm clearly had a high transcript level in the thorax where the prothoracic glands were located. Dietary introduction of double-stranded RNA （dsRNA） of LsPhm at the nymph stage successfully knocked down the target gene, decreased expres- sion level ofecdysone receptor （LsEcR） gene and caused a higher nymphal mortality rate and delayed development. Ingestion of 20-hydroxyecdysone on LsPhm-dsRNA-exposed nymphs did not increase LsPhm expression level, but almost completely rescued the LsEcR mRNA level, and relieved the negative effects on survival and development. Thus, our data suggest that the putative LsPhm encodes a ftmctional 25-hydroxylase that catalyzes the biosynthesis of ecdysteroids in L. striatellus.

Laodelphax striatellus Atg8 facilitates Rice stripe virus infection in an autophagy-independent manner

Rice stripe virus(RSV)is the causative agent of rice stripe disease and is completely dependent on insect vectors for its plant-to-plant transmission.Laodelphax striatellus is the major insect vector for RSV.In this study,we explored the interactions be-tween RSV infection and L.striatellus autophagy,a potential intrinsic antiviral mechanism in insects.We found that L.striatellus autophagic activity did not affect RSV infection;however,the autophagy related-8(Atg8)gene significantly enhanced virus infection.Dur-ing RSV initial infection within the L.striatellus midgut,silencing of Atg8 expression significantly decreased the phosphorylation of c-Jun N-terminal kinase(p-JNK);however,when RSV infection is absent,silencing of Atg8 did not alter p-JNK levels.Thesc results indicated that Atg8 might activate the JNK machinery by allowing more virus infection into cells.We further revealed that Atg8-deficiency significantly decreased RSV accumu-lation on the surface of the insect midgut epithelial cells,suggesting a receptor trafficking function of the y-aminobutyric acid receptor-associated protein family.Using the RSV ovary entry as a model,in which vitellogenin receptor(V gR)mediates RSV cell entry,we clarified that Atg8-deficiency decreased the abundance of V gR localizing on the cytomem-brane and disturbed the attachment of RSV in the germarium zones.Collectively,these results revealed an autophagy-independent function of L.striatellus Atg8 that enhances RSV initial infection by increasing virus attachment on the infection sites.

NDUFA8 potentially rescues Wolbachia-induced cytoplasmic incompatibility in Laodelphax striatellus

The endosymbiont Wolbachia manipulates host reproduction by several strategies, one of the most important of which is cytoplasmic incompatibility (CI). CI can be rescued when Wolbachia-infected males mate with females infected with the same Wolbachia strain. However, the potential rescue mechanism of CI in the small brown planthopper Laodelphax striatellus is unclear. In this study, comparative transcriptome analysis was applied to explore the effect of Wolbachia on L. striatellus eggs. A total of 1387 differentially expressed genes were identified. RNA interference of 7 Wolbachia-upregulated key planthopper genes reduced egg reproduction, suggesting that Wolbachia might improve fecundity in L. striatellus by affecting these 7 genes. Suppressing the expression of another upregulated gene, NDUFA8 (encoding NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 8-like) by RNA interference significantly increased the mortality of early embryos without affecting the number of deposited eggs. Wolbachia infection upregulated the mRNA level of NDUFA8, and dsNDUFA8 treatment of Wolbachia-infected females recreated CI-like symptoms, suggesting that NDUFA8 is associated with the rescue phenotype. Because all L. striatellus populations worldwide are infected with Wolbachia, NDUFA8 is a potential pest control target.

High temperature determines the ups and downs of small brown planthopper Laodelphax striatellus population

Small brown planthopper, Laodelphax striatellus （Fall6n） numbers usually drop sharply in the summer and revive quickly in the autumn. However, it is unclear whether and how the high temperature plays a role in this process. The effects of durations of heat exposure （33~C） on life-history traits were examined here. Exposure of adults for 1 day during the oviposition stage led to a very low survival of nymphs. The average longevity ofL. striatellus exposed for 1-31 days from oviposition was significantly longer than that of the control （27℃）. Short-term （1-5 days） heat exposure of the third instar nymphs did not significantly influence eclosion, but exposure of the fourth instar nymphs significantly increased eclosion. Lifespan from egg to adult was significantly lengthened when the third instar nymphs were exposed to heat for 2-15 days, or the fourth instar were exposed for 10 days. The preoviposition period was prolonged by heat exposure of the third or fourth instar nymphs. Short-term heat exposure of less than 3 days of the third or fourth instar nymphs did not restrict fecundity, but when the exposure duration exceeded 5 days the total eggs per female and hatchability decreased. Exposure to high temperature increased the brachypter rate of adults. In summary, low survival and slowing development under heat exposure resulted in population decline in the summer, and the relatively high fecundity and brachypter rate led to quick revival in autumn. Temperature in the summer determines the rise and fall in numbers ofL. striatellus.

Identification of a fatty acid synthase gene(FAS1)from Laodelphax striatellus planthoppers contributing to fecundity

Fatty acid synthase (FAS) is a multifunctional enzyme that plays an important role in the formation of fatty acids. The fatty acids take part in many processes, such as cell signaling and energy metabolism, and in insects they are important in both cuticular hydrocarbon (CHC) formation and reproduction. Here we characterized the sequence structure and function of an FAS from the small brown planthopper (SBPH), Laodelphax striatellus. The full-length open reading frame (ORF) sequence of LsFAS1 was 7122 bp, encoding a predicted protein of 2373 amino acid residues. There were 7 functional domains in the LsFAS1 protein sequence. Gene expression screening by real-time quantitative polymerase chain reaction (RT-qPCR) showed that LsFAS1 was expressed in all developmental stages. Relative expression was highest at the 4th-instar and female adult stages. Among different tissues, the expression level of LsFAS1 in the ovary was the highest. Phylogenetic analysis showed that LsFAS1 clustered in a clade with 2 FASs from Nilaparvata lugens. Furthermore, these 3 FASs are related to cockroach BgFAS and locust LmFAS. After RNA interference-mediated knock-down, most treated insects died at eclosion. In addition, the lifespan of dsFAS1-treated female adults was shorter than that of the dsGFP-injected control, and offspring production decreased. Also, the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes decreased. Virgin females dissected at days 2 and 4 post-eclosion showed many matured oocytes in planthoppers treated with dsGFP but not with dsFAS1. These data highlight the importance of LsFAS1 in SBPH, including a role in reproduction.

水稻播种期对灰飞虱及其传播的条纹叶枯病发生流行的影响

【目的】了解水稻播种期对灰飞虱及其传播的条纹叶枯病发生流行的影响。【方法】2006～2007年2年田间设置4个播种期,调查分析水稻灰飞虱种群动态和条纹叶枯病发病的程度。【结果】播种期是影响条纹叶枯病的重要因子。在浙江北部,单季晚稻随着播种期的推迟,病情递减;秧田期株发病率与播种期的关系可以Weibull方程来描述、与秧田期灰飞虱种群动态曲线下面积(AUCPD,即累计虫日)或高峰期密度则呈Logistic关系;本田稳定期发病率则可以以播种期、秧田期灰飞虱高峰期密度、秧田末期的株发病率来描述,而本田期的AUCPD并不是描述该阶段的发病率的必要变量。【结论】在综合治理中,适期播种是控制该病害流行的最有效方法之一,在浙江北部单季粳稻移栽的适宜播种期为5月底～6月上旬。

小麦开花灌浆初期喷施农药对灌浆后期灰飞虱的影响及生化分析

为明确麦田常规使用农药对灌浆后期灰飞虱虫量的影响并揭示原因,为稻麦病虫害的综合治理提供理论依据,通过大田试验,开花初期喷施杀菌剂,花后7d喷施杀虫剂,研究了小麦花后5种农药(杀菌剂:多菌灵、咪鲜胺;杀虫剂:毒死蜱、乐果和吡虫啉)对小麦灌浆后期灰飞虱的影响。结果表明,施用多菌灵、毒死蜱和乐果均能导致小麦灌浆后期灰飞虱虫口密度显著增加,其中毒死蜱和毒死蜱加多菌灵处理分别使虫量增加255.2%和425.6%。花后27d测定小麦叶、穗中的游离氨基酸、还原糖、酚含量和蔗糖转化酶(Invertase,Inv)、多酚氧化酶(Polyphenol oxidase,PPO)活性,结果显示,与对照相比,除吡虫啉和咪鲜胺外的农药处理均使小麦叶、穗中的游离氨基酸、还原糖含量和Inv活性增加,酚含量和PPO活性降低,以多菌灵加毒死蜱处理的调控效应最大。经相关分析,麦田灰飞虱虫口密度与小麦叶、穗中游离氨基酸含量、还原糖含量和Inv活性呈正相关,与小麦叶中酚含量和PPO活性呈负相关,说明农药对灰飞虱虫量的影响,可能是由于农药使小麦植株生理生化发生变化而引起。把灰飞虱虫量作为重要考虑因素,建议防治小麦赤霉病时宜轮换使用多菌灵与咪鲜胺,防治麦蚜时宜选用吡虫啉。

Effect of Rice Sowing Date on Occurrence of Small Brown Planthopper and Epidemics of Planthopper-Transmitted Rice Stripe Viral Disease

To understand the relationship between rice sowing date and occurrence of the rice small brown planthopper （SBPH） Laodelphax striatellus Fallen and the epidemics of the planthopper-transmitted rice stripe viral （RSV） disease, four sowing dates of rice were evaluated in 2006 and 2007. The results showed that the peak density of SBPH and RSV incidence in the nursery and in the transplanted field decreased with the delay of sowing date in single crop of japonica rice in north Zhejiang Province of China. The relationship between seedling RSV incidence at the end of the nursery trial with sowing date was well described by Weibull equation. The area under the curve of population dynamics （AUCPD or planthopper-day accumulation） or the peak density of the planthopper in the nursery could be summarized by a logistic equation. RSV incidence in the transplanted fields could be characterized quantitatively by a multivariate regression equation, including the variables of sowing date, peak density of the vector, and RSV incidence at the end of the nursery trial. That the descriptive model excluded the AUCPD in transplanted field implies that this variable is not necessary in forecasting disease epidemics in the field. The 2-year experiments sufficiently indicated that suitable sowing of rice could be used as one of the effective measures to control the vector population and therefore the planthopper-transmitted RSV on a larger scale. The optimal sowing date for the single-cropped transplanted japonica rice is recommended from late May to early June in north Zhejiang, China.

The Empidemiology of Wheat Rosette Stunt in Hulunbeir District of Inner-Mongolia

The virus disease threatening wheat production in Hulunbcir District of Inner - Mongolia was identified as NCMV (Northern Cereal Mosaic Virus) or WRSV (Wheat Rosette Stunt Virus) by tests of host range, transmission, virus particle and scrology. The main vector for spreading the virus is planthopper, Laodelphax striatellus (Fallen), which overwinters in the stubbles of wheat underground. The overwintered virulifcrous nymphs emerged in late April is responsible for the early infection of the disease. Agropyron repens, an important perennial weed in cultivated regions, is also an important wild host of the virus and its vector. Severe loss is induced when wheat is infected before 3 - leaf stage. The disease incidence could be predicted by a model with the population and virulifcrous rate of overwintered vectors as independent variables.

Similarities and spatial variations of bacterial and fungal communities in field rice planthopper (Hemiptera: Delphacidae) populations

Rice planthoppers are notorious plant sap-feeding pests which cause serious damage.While several microbes in rice planthoppers have been broadly characterized,the abundance and diversity of bacteria and fungi in field planthoppers are largely unknown.This study investigated the bacterial and fungal community compositions of Chinese wild rice planthoppers Laodelphax striatellus and Sogatella furcifera using parallel 16S rRNA gene amplicon and internal transcribed space region sequencing.The bacteria varied sig-nificantly between the species and were partitioned significantly by sex,tissues and host environments in each species.The majority of bacteria were affiliated with the genera Wol-bachia,Cardinium,Rickettsia and Pantoea.The abundance of Wolbachia was negatively correlated with that of Cardinium in both planthopper species.Compared with bacteria,the abundance and diversity of fiungi did not differ between sexes but both were enriched in the gut.The bacterial community as a whole showed no significant correlation with the fungal community.The majority of fungi were related to Sarocldium,Alternaria,Malasseria,Aspergillus and Curvularia.A phylogenetic analysis revealed that these fungi were closely related to botanic symbionts or pathogens.Our results provide novel insights into the bacteria and fungi of rice planthoppers.