The evolution and functional divergence of 10 Apolipoprotein D-like genes in Nilaparvata lugens

Apolipoprotein D(ApoD),a member of the lipocalin superfamily of proteins,is involved in lipid transport and stress resistance.Whereas only a single copy of the ApoD gene is found in humans and some other vertebrates,there are typically several ApoD-like genes in insects.To date,there have been relatively few studies that have examined the evolution and functional differentiation of ApoD-like genes in insects,particularly hemi-metabolous insects.In this study,we identified 10 ApoD-like genes(NlApoD1−10)with distinct spatiotemporal expression patterns in Nilaparvata lugens(BPH),which is an important pest of rice.NlApoD1−10 were found to be distributed on 3 chromosomes in a tandem array of NlApoD1/2,NlApoD3−5,and NlApoD7/8,and show sequence and gene structural divergence in the coding regions,indicating that multiple gene duplication events occurred during evolution.Phylogenetic analysis revealed that NlApoD1−10 can be clustered into 5 clades,with NlApoD3−5 and NlApoD7/8 potentially evolving exclusively in the Delphacidae family.Functional screening using an RNA interference approach revealed that only NlApoD2 was essential for BPH development and survival,whereas NlApoD4/5 are highly expressed in testes,and might play roles in reproduction.Moreover,stress response analysis revealed that NlApoD3−5/9,NlApoD3−5,and NlApoD9 were up-regulated after treatment with lipopolysaccharide,H2O2,and ultraviolet-C,respectively,indicating their potential roles in stress resistance.

Expression of two types of acetylcholinesterase gene from the silkworm, Bombyx mori, in insect cells

Complementary DNAs encoding two types of acetylcholinesterase （ACHE） were isolated from the silkworm, Bombyx mori. The type 1 （Bmacel） and type 2 （Bmace2） ORFs are 2052 and 1917 bp in length, respectively. Both the complete ORFs of the Bmaces and C- terminal truncated forms were recombined into the Bacmid baculovirus vector under the control of the polyhedrin promoter and expressed in Trichoplusia ni （Tn-5B 1-4） cells. The resulting products exhibited ACHE activity and glycosylation of the expressed proteins. An inhibition assay indicated that the ace2-type enzyme was more sensitive than the acel-type enzyme to inhibition by eserine and paraoxon.

FAR gene enables the brown planthopper to walk and jump on water in paddy field

Many insects can live on water and survive being caught in the rain.Current research has shown that insect cuticular hydrocarbons(CHC)confer desiccation resistance to maintain water balance.In this study,we identified a fatty acyl-CoA reductase gene(NlFAR)of the rice brown planthopper,Nilaparvata lugens that is essential for the production of CHCs,and found that NlFAR is essential for N.lugens to walk and jump on water when moving from one rice plant to another in paddy fields.NlFAR was mainly expressed in the integument at the beginning of each molt.Cuticular surface analysis by scanning electron microscopy and characterization of CHC extracts indicated that N.lugens with knockdown of NlFAR using RNA inference(RNAi)had a neater epicuticle layer and a significant decrease in CHC contents.Knockdown of NlFAR did not influence the desiccation resistance of N.lugens,but the ds NlFAR-treated insects were easily adhered and moistened by water droplets or their own secreted honeydew and unable to walk or jump on water.These results suggested that NlFAR is a crucial enzyme for CHC biosynthesis and cuticle waterproofing,but not for water retention of N.lugens,which may provide a potential strategy for pest management.

Characteristics of the draft genome of ＂Candidatus Arsenophonus nilaparvatae＂, a facultative endosymbiont of Nilaparvata lugens

There exists a kind of symbiotic bacterium named ＂Candidatus Arsenophonus nilaparvatae＂ in the brown planthopper （BPH）, Nilaparvata lugens. After being filtered and assembled from the BPH genome sequencing project, the genome sequence of this bacterial symbiont was obtained. After initial analysis based on the genome, we have found its potential role to synthesize B vitamins for the host. In order to better understand the lifestyle and the genomic changes of this symbiotic bacterium after the symbiotic relationship was established, we further report the characteristics of this draft genome. Compared with several other related bacteria, ＂Candidatus Arsenophonus nilaparvatae＂ has proven to be a facultative endosymbiont at the genomic level. Concurrently, the presence of fimbriae and flagella formation related genes indicates this maternally transmitted endosymbiont is most likely to retain the capacity to invade new hosts. Through further analysis of annotated gene sets, we also find evidence of genome reduction in its secretion system and metabolic pathways. These findings reflect its evolutionary trend to be an obligate one and enable a deeper study of microbe-insect interactions.

Chitin synthase 1 and five cuticle protein genes are involved in serosal cuticle formation during early embryogenesis to enhance eggshells in Nilaparvata lugens

Many holo-and hemimetabolous insects enhance their eggshells during embryogenesis by forming a serosal cuticle(SC).To date,scholarly understanding of the SC composition and SC-related gene functions has been limited,especially for hemimetabolous insects.In this study,we initially performed transmission electron microscopic(TEM)observation and chitin staining of the SC in Nilaparvata lugens,a hemimetabolous rice pest known as the brown planthopper(BPH).We confirmed that the SC was a chitin-rich lamellar structure deposited gradually during the early embryogenesis.Parental RNA interference(RNAi)against Nilaparvata lugens chitin synthase 1(NlCHS1)in newly emerged and matured females resulted in decreases of egg hatchability by 100%and 76%,respectively.Ultrastructural analyses revealed loss of the lamellar structure of the SC in dsNlCHS1-treated eggs.According to temporal expression profiles,five cuticle protein coding genes,NlugCpr1/2/3/8/90,were specifically or highly expressed during the SC formation period,and NlugCpr1/2/3/90 were further detected in 72 h eggshells extract by ultra-performance liquid chromatography-tandem mass spectrometry/mass spectrometry.NlugCpr2/3/90 were likely three SC-specific cuticle proteins.TEM observations of the SC following parental RNAi against NlugCpr1/2/3/8/90 demonstrated that NlugCpr3/8/90 were essential for SC formation.The study provided an understanding of the SC formation process and SC-related cuticle proteins in BPHs,which offer potential targets for pest control in the egg stage as well.

Identification of salivary proteins in the whitefly Bemisia tabaci by transcriptomic and LC-MS/MS analyses

The whitefly Bemisia tabaci is a notorious agricultural pest of many crops worldwide.Although it is thought that B.tabaci secretes saliva into the host plant to counter plant defenses,knowledge on the whitefly salivary proteome is limited.Here,we characterized the gene/protein repertoires of B.tabaci salivary glands and secreted saliva by transcriptomic and liquid chromatography tandem mass spectroscopy analyses.A total of 698 salivary gland-enriched unigenes and 171 salivary proteins were identified.Comparative analysis between the B.tabaci salivary proteins and those of different arthropod species revealed numerous similarities in proteins associated with binding,hydrolysis,and oxidation-reduction,which demonstrates a degree of conservation across herbivorous saliva.There were 74 proteins only identified in B.tabaci saliva,of which 34 were B.tabaci-specific.In addition,13 salivary proteins,of which I1 were B.tabaci-specific,were differentially regulated when B.tabaci fed on different hosts.Our results provide a good resource for future functional studies of whitefly salivary effectors,and might be useful in pest management.

Analysis of a late gene, orfl01 from Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirns open reading frame 101 （ha101） is 762 nts in length and encodes a 254 amino acid peptide with predicted 29 kDa molecular weight. The homologues of ha101 were explored using BLASTP searching tool in the updated GenBank/EMBL and SWlSS-PROT databases. The results showed that the homologues of ha101 were present in all the completely sequenced lepidopteran nucleopolyhedrovirnses and granulovirnses, suggesting that ha101 might be a functional gene associated with their lepidopteran hosts. Sequence alignment of ha101 and its homologues revealed that 10 amino acids were completely conserved. RT-PCR analysis of ha101 manifested that the transcript of ha101 was first detected at 24 hpi and remained detectable at up to 122 hpi, suggesting that ha101 was transcribed during late stages of infection. Ha101 was expressed using Bac to Bac system in Tn5B-1-4 cells. The product of ha101 expressed in Tn5B-1-4 cells was approximately 29kDa, consistent with the predicted molecular weight, and the results were confirmed by western blot analysis. The subcellular localization indicated that ha101 was aggregated along nuclear envelope during the early stages of infection and spread out to the entire nucleus including virogenic stroma in late stages of infection, suggesting that ha101 may play a specific role in virion assembly process or virogenic stroma arrangement.

Three-dimensional reconstruction of pore canals in the cuticle of the brown planthopper

Dear Editor,The insect cuticle is a multifunctional coat that defines and stabilizes the shape of the body,appendages and internal organs,serves as a barrier against water and chemical compounds,such as insecticides,prevents infection and protects against predators(Moussian,2010).The surface of insects is covered by a lipid layer composed of diverse hydrocarbons,wax esters,fatty acids,fatty alcohols,sterols and triglycerides.Delivery of lipids to the body surface occurs through the cuticle by a nano-canal system consisting of pore canals(PC)and wax canals(WC).

Using chimeric piggyBac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells

The piggyBac transposon has been long used to integrate foreign DNA into insect genomes.However,undesirable transgene expression can result from random insertions into the genome.In this study,the efficiency of chimeric Gal4-piggyBac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2(S2) cells.The Gal4-piggyBac transposase has a Gal4 DNA-binding domain(DBD),and the target plasmid has upstream activating sequences(UAS) to which the Gal4 DBD can bind with high affinity.The results indicate that,in the Bm-12 and S2 cells,transpositional activity of Gal4-piggyBac transposase was increased by 4.0 and 7.5 times,respectively,compared to controls,where Gal4-UAS interaction was absent.Moreover,the Gal4-piggyBac transposase had the ability of directing piggyBac element integration to certain sites of the target plasmid,although the target-directing specificity was not as high as expected.The chimeric piggyBac transposase has the potential for use in site-directed transgenesis and gene function research in B.mori.

A draft genome of the ghost moth, Thitarodes （Hepialus） sp., a medicinal caterpillar fungus

Dear Editor, The Chinese caterpillar fungus, commonly known as ＂Dong Chong Xia Cao＂ （winter worm summer grass） in China, is one of the most valuable medicinal fungi in the world （Zhang et al., 2012）. In the alpine meadows of the Tibetan Plateau （altitude 3500-5000 m）, the ascomycete Ophiocordyceps sinensis （syn. Cordyceps sinensis） par- asitizes the larvae of the ghost moth （Thitarodes spp.）, forming a fungus-caterpillar complex. The caterpillar fun- gus has long been used in traditional Chinese medicine （TCM） for hundreds of years, with a diverse list of phar- macological effects （Zhu et al., 1998）.