Molecular characterization,spatial‐temporal expression and magnetic response patterns of iron‐sulfur cluster assembly1 (IscA1) in the rice planthopper,Nilaparvata lugens

The mechanisms of magnetoreception have been proposed as the magnetitebased, the chemical radical-pair and biocompass model, in which magnetite particles, the cryptochrome (Cry) or iron-sulfur cluster assembly 1 (IscA1) may be involved. However, little is known about the association among the molecules. Here we investigated the molecular characterization and the mRNA expression of IscA1 in different developmental stages, tissues and magnetic fields in the migratory brown planthopper (BPH), Nilaparvata lugens. NlIscA1 contains an open reading frame of 390 bp, encoding amino acids of 129, with the predicted molecular weight of 14.0 kDa and the isoelectric point of 9.10. Well-conserved Fe-S cluster binding sites were observed in the predicted protein. Phylogenetic analysis demonstrated NlIscA1 to be clustered into the insect's IscA1. NlIscA1 showed up-regulated mRNA expression during the period of migration. The mRNA expression of NlIscA1 could be detected in all the three tissues of head, thorax and abdomen, with the highest expression level in the abdomen. For the macropterous migratory Nilaparvata lugens, mRNA expression of NlIscA1 and N. lugens cryptochromel (Nlcry1) were up-regulated under the magnetic fields of 5 Gauss and 10 Gauss in strength (vs. local geomagnetic field), while N. lugens cryptochrome 2 (Nlcry2) remained stable. For the brachyterous non-migratory Nilaparvata lugens, no significant changes were found in mRNA expression of NlIscA1, Nlcry1 and Nlcry2 among different magnetic fields. These findings preliminarily reveal that the expression of NlIscA1 and Nlcry1 exhibited coordinated responses to the magnetic field. It suggests some potential associations among the putative magneto-sensitive molecules of cryptochrome and iron-sulfur cluster assembly.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

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.     

Tomato CRY1a plays a critical role in the regulation of phytohormone homeostasis,plant development,and carotenoid metabolism in fruits

Blue light photoreceptors, cryptochromes (CRYs), regulate multiple aspects of plant growth and development. However, our knowledge of CRYs is predominantly based on model plant Arabidopsis at early growth stage. In this study, we elucidated functions of CRY1a gene in mature tomato (Solanum lycopersicum) plants by using cry1a mutants and CRY1a‐overexpressing lines (OE‐CRY1a‐1 and OE‐CRY1a‐2). In comparison with wild‐type plants, cry1a mutants are relatively tall, accumulate low biomass, and bear more fruits, whereas OE‐CRY1a plants are short stature, and they not only flower lately but also bear less fruits. RNA‐seq, qRT‐PCR, and LC‐MS/MS analysis revealed that biosynthesis of gibberellin, cytokinin, and jasmonic acid was down‐regulated by CRY1a. Furthermore, DNA replication was drastically inhibited in leaves of OE‐CRY1a lines, but promoted in cry1a mutants with concomitant changes in the expression of cell cycle genes. However, CRY1a positively regulated levels of soluble sugars, phytofluene, phytoene, lycopene, and ß‐carotene in the fruits. The results indicate the important role of CRY1a in plant growth and have implications for molecular interventions of CRY1a aimed at improving agronomic traits.     

Characterization of a Nilaparvata lugens (Stål) brummer gene and analysis of its role in lipid metabolism

The brummer (bmm) genes encode the lipid storage droplet‐associated triacylglycerols (TAG) lipases, which belong to the Brummer/Nutrin subfamily. These enzymes hydrolyze the ester bonds in TAG in lipid metabolism and act in insect energy homeostasis. Exposure to some agricultural chemicals leads to increased fecundity, which necessarily involves lipid metabolism, in some planthopper species. However, the biological roles of bmm in planthopper lipid storage and mobilization have not been investigated. Here, the open reading frame (ORF) of bmm (Nlbmm) was cloned and sequenced from the brown planthopper (BPH; Nilaparvata lugens). The ORF is 1014 bp encoding 338 amino acid residues. Nlbmm contained patatin domains and shared considerable evolutionary conservation with other insect bmms. Nlbmm is highly expressed in the fat body, consistent with its roles in lipid metabolism. Injection with Nlbmm double‐stranded RNA (dsNlbmm) led to reduced Nlbmm mRNA accumulation, but did not influence expression of several genes related to lipid synthesis including acyl‐CoA‐binding protein (ACBP), acetyl‐CoA carboxylase (ACC), and a lipophorin receptor (LpR). Nlbmm knockdown led to increased TAG contents in whole bodies, accumulation of total fat body lipid, and decreased hemolymph lipid content. Nlbmm knockdown did not influence the synthesis and distribution of glycerol. We infer that Nlbmm acts in TAG breakdown and fat metabolism in N. lugens.     

Cloning,expression profiling,and acetylation identification of alpha‐tubulin N‐acetyltransferase 1 from Bombyx mori

Alpha‐tubulin N‐acetyltransferase 1 (ATAT1) is an acetyltransferase specific to α‐tubulin and performs important functions in many cellular processes. Bombyx mori is an economic insect and also known as a model lepidoptera insect. In this study, we cloned a B. mori ATAT1 gene (BmATAT1) (Gen Bank accession number: XP_004932777.1). BmATAT1 contained an open reading frame (ORF) of 1,065 bp encoding 355 amino acids (aa). Expression profiling of BmATAT1 protein showed that the expression levels of BmATAT1 at different developmental stages and different tissues in fifth‐instar larvae differ. BmATAT1 was highly expressed at the egg stage and in the head of the fifth‐instar larvae. Subcellular localization showed that BmATAT1 was distributed in the cytoplasm and nucleus. Furthermore, BmATAT1 may lead to time‐dependent induction of cell cycle arrest in the G2/M phase by flow cytometry analysis. Interestingly, using site‐specific mutation, immunoprecipitation, and Western blotting, we further found a BmATAT1 acetylated site at K156, suggesting that this acetyltransferase could be regulated by acetylation itself.     

Cultivation,identification and quantification of one species of yeast‐like symbiotes,Candida, in the rice brown planthopper,Nilaparvata lugens

Abstract The brown planthopper (BPH), Nilaparvata lugens Stål, which is one of the most destructive pests of rice, has been confirmed to harbor yeast‐like symbiotes (YLS) in the fat body. Several morphologically different YLS have been previously isolated and cultured in vitro from BPH, but direct evidence is lacking to further clarify whether the cultured YLS were from BPH. In this study, one species of YLS was successfully cultured in vitro and simultaneously verified to exist in the fat body of BPH by sequence analysis and nested polymerase chain reaction (PCR). The cultured YLS isolate in vitro was identified as a member of the genus Candida on the basis of 18S rDNA (ribosomal DNA) and 5.8S‐ITS (internal transcribed spacer) rDNA sequence and a phylogenetic analysis of ITS sequences from yeast. Therefore, this yeast isolate was named as Candida‐like symbiotes. Candida‐like symbiotes was found to exist in fat bodies, ovaries and newly laid eggs of the BPH, but not in the heads, thoraxes and mid‐guts. In addition, the number of Candida‐like symbiotes in 1 × 10⁶ of purified YLS from BPH fat bodies was speculated to be (5.32 ± 0.22) × 10⁴ on the basis of a quantitative PCR analysis.     

Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against brown planthopper (Nilaparvata lugens) in transgenic rices

Plant defensins are small (5-10 kDa) basic peptides thought to be an important component of the defense pathway against fungal and/or bacterial pathogens. To understand the role of plant defensins in protecting plants against the brown planthopper, a type of insect herbivore, we isolated the Brassica rapa Defensin 1 (BrD1) gene and introduced it into rice (Oryza sativa L.) to produce stable transgenic plants. The BrD1 protein is homologous to other plant defensins and contains both an N-terminal endoplasmic reticulum signal sequence and a defensin domain, which are highly conserved in all plant defensins. Based on a phylogenetic analysis of the defensin domain of various plant defensins, we established that BrD1 belongs to a distinct subgroup of plant defensins. Relative to the wild type, transgenic rices expressing BrD1 exhibit strong resistance to brown planthopper nymphs and female adults. These results suggest that BrD1 exhibits insecticidal activity, and might be useful for developing cereal crop plants resistant to sap-sucking insects, such as the brown planthopper.     

Phylogeny and oscillating expression of period and cryptochrome in short and long photoperiods suggest a conserved function in Nasonia vitripennis

Photoperiodism, the ability to respond to seasonal varying day length with suitable life history changes, is a common trait in organisms that live in temperate regions. In most studied organisms, the circadian system appears to be the basis for photoperiodic time measurement. In insects this is still controversial: while some data indicate that the circadian system is causally involved in photoperiodism, others suggest that it may have a marginal or indirect role. Resonance experiments in the parasitic wasp Nasonia vitripennis have revealed a circadian component in photoperiodic time measurement compatible with a mechanism of internal coincidence where a two components oscillator system obtains information from dawn and dusk, respectively. The identity of this oscillator (or oscillators) is still unclear but possible candidates are the oscillating molecules of the auto-regulatory feedback loops in the heart of the circadian system. Here, we show for the first time the circadian oscillation of period and cryptochrome mRNAs in the heads of Nasonia females kept under short and long photoperiods. Period and cryptochrome mRNA levels display a synchronous oscillation in all conditions tested and persist, albeit with reduced amplitude, during the first day in constant light as well as constant darkness. More importantly, the signal for the period and cryptochrome oscillations is set by the light-on signal. These results, together with phylogenetic analyses, indicate that Nasonia’s period and cryptochrome display characteristics of homologous genes in other hymenopteran species.     

The role of chemosensory protein 10 in the detection of behaviorally active compounds in brown planthopper,Nilaparvata lugens

Chemosensory proteins (CSPs) play important roles in insects’ chemoreception, although their specific functional roles have not been fully elucidated. In this study, we conducted the developmental expression patterns and competitive binding assay as well as knock‐down assay by RNA interference both in vitro and in vivo to reveal the function of NlugCSP10 from the brown planthopper (BPH), Nilaparvata lugens (Stål), a major pest in rice plants. The results showed that NlugCSP10 messenger RNA was significantly higher in males than in females and correlated to gender, development and wing forms. The fluorescence binding assays revealed that NlugCSP10 exhibited the highest binding affinity with cis‐3‐hexenyl acetate, eicosane, and (+)‐β‐pinene. Behavioral assay revealed that eicosane displayed attractant activity, while cis‐3‐hexenyl acetate, similar to (+)‐β‐pinene significantly repelled N. lugens adults. Silencing of NlugCSP10, which is responsible for cis‐3‐hexenyl acetate binding, significantly disrupted cis‐3‐hexenyl acetate communication. Overall, findings of the present study showed that NlugCSP10 could selectively interrelate with numerous volatiles emitted from host plants and these ligands could be designated to develop slow‐release mediators that attract/repel N. lugens and subsequently improve the exploration of plans to control this insect pest.     

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.     

褐飞虱ABCB基因的克隆与表达分析

三磷酸腺苷结合盒转运蛋白(ATP-binding cassette,ABC)广泛分布于各种生物体中,是最大的膜蛋白家族之一。在昆虫中,ABC转运子不仅在分子转运过程中有重要的功能,同时在杀虫剂抗性、代谢和发育中都起到重要的作用。本研究克隆和鉴定了褐飞虱Nilaparvata lugens St l ABCB基因(NlABCB),结果表明NlABCB基因全长1468 bp,含有一个长度为459 bp的开放阅读框,编码153个氨基酸。利用定量PCR检测NlABCB基因在褐飞虱体内的表达,发现NlABCB转录本在褐飞虱的所有发育阶段都有表达,在1龄若虫中表达量最低,随着生长发育表达量逐渐升高。NlABCB在褐飞虱5龄若虫的中肠中表达量最高。研究发现该基因与褐飞虱的生理活动有关。构建NlABCB-dsRNA载体,转化大肠杆菌Escherichia coli HT115后,经IPTG诱导,形成NlABCB-dsRNA,喂养褐飞虱取食含NlABCB-dsRNA人工饲料,发现褐飞虱的存活率明显降低。因此该基因的克隆和功能鉴定,对于作物的抗虫育种,尤其对水稻害虫的生物防治将起到重要的促进作用。