管纹艳虎天牛UDP-葡萄糖基转移酶基因的鉴定及表达特征分析

【目的】昆虫UDP-葡萄糖基转移酶(UGT)在其内源性和外源性有毒化合物的解毒代谢过程中起着重要作用,但在管纹艳虎天牛Rhaphuma horsfieldi中UGT基因的研究尚属空白。【方法】采用转录组学、生物信息学、进化和表达谱分析、蛋白同源建模等技术研究了管纹艳虎天牛的UGT基因家族。【结果】从管纹艳虎天牛转录组中一共鉴定到36个RhorUGTs基因,其中17个具有全长序列。鞘翅目不同种间UGT基因数量的比较表明,管纹艳虎天牛具有中等数量的UGT基因。进化分析结果表明,RhorUGTs分布在10个亚家族中,其中UGT352亚家族为天牛科昆虫所特有,且该亚家族中RhorUGT2/7/10/16/18/27可能通过基因的复制产生。表达谱结果表明,大部分RhorUGTs基因在检测的所有组织中均有表达,部分基因呈现触角或跗节特异或高表达的特点,暗示它们具有嗅觉或触觉等功能。三级结构分析发现,RhorUGT17的α3、α4、β4和β5主要参与UDP-葡萄糖的结合。【结论】本研究明确了管纹艳虎天牛UGT基因的数量、序列特征、进化关系及组织表达特征,研究结果为该种天牛解毒代谢机制的阐明奠定基础。     

管纹艳虎天牛糖苷水解酶基因家族的鉴定和表达谱分析

昆虫糖苷水解酶（Glycoside hydrolase, GH）在寄主植物糖类化合物的水解过程中扮演着重要作用,但是在管纹艳虎天牛Rhaphuma horsfieldi中尚未有GH基因的报道。基于测序的转录组数据,本研究从管纹艳虎天牛中鉴定到7个RhorGHs家族：GH1、GH9、GH13、GH16、GH28、GH31和GH45,分别具有23、1、4、2、10、12和4个基因,其中23个基因具有全长序列。在进化分析中,管纹艳虎天牛不同GH家族的成员主要以物种特异的形式聚类,尤其是GH1和GH28家族。三级结构分析显示,RhorGH28家族的蛋白主要由β折叠组成,不同蛋白间具有高度保守的结构和与糖类化合物互作的关键氨基酸位点,包括质子供体、催化亲核残基和底物结合位点。在表达谱分析中,大部分RhorGHs基因主要在雌雄虫腹部特异或高表达,暗示其可能在与消化有关的肠道中表达。研究结果明确了管纹艳虎天牛GH家族及其数量,以及各家族的序列特征和进化关系,可为该天牛的寄主植物适应性机制研究提供借鉴。     

异色跗虎天牛的种内变异探讨及再描述(鞘翅目,天牛科,天牛亚科)

对2011年6月采自云南勐仑的73头异色跗虎天牛Perissus mutabilis Gahan,1894标本进行研究,发现该种73号标本在鞘翅斑纹、前足爪开式、前胸背板颜色及斑点、后足腿节长度存在不同程度的差异,研究认为上述特征不宜用作该种分类鉴定的特征。红胸跗虎天牛Perissus mutabilis vitabilis Pic,1923和黑胸跗虎天牛Perissus mutabilis obscuricolorPic,1937依据部分上述特征建立,且特征出现交叉,分布区域与指名亚种异色跗虎天牛P.mutabilis mutabilis Gahan,1894重叠,建议取消红胸跗虎天牛P.mutabilis vitabilis Pic,1923和黑胸跗虎天牛P.mutabilis obscuricolor Pic,19372个亚种。并对异色跗虎天牛P.mutabilis Gahan,1894进行重描述。     

A REDESCRIPTION OF PERISSUS MUTABILIS GAHAN (COLEOPTERA, CERAMBYCIDAE, CERAMBYCINAE) AND DISCUSSION OF THE INTRASPECIFIC VARIATION

对2011年6月采自云南勐仑的73头异色跗虎天牛Perissus mutabilis Gahan,1894标本进行研究,发现该种73号标本在鞘翅斑纹、前足爪开式、前胸背板颜色及斑点、后足腿节长度存在不同程度的差异,研究认为上述特征不宜用作该种分类鉴定的特征.红胸跗虎天牛Perissus mutabilis vitabilis Pic,1923和黑胸跗虎天牛Perissus mutabilis obscuricolor Pic,1937依据部分上述特征建立,且特征出现交叉,分布区域与指名亚种异色跗虎天牛P.mutabilis mutabilis Gahan,1894重叠,建议取消红胸跗虎天牛P.l mutabilis vitabilis Pic,1923和黑胸跗虎天牛P.mutabilis obscuricolor Pic,1937 2个亚种.并对异色跗虎天牛P.mutabilis Gahan,1894进行重描述.     

虎天牛族中国六新纪录种(鞘翅目,天牛科,天牛亚科)

共记述了绿虎天牛属Chlorophorus Chevrolat中国1新纪录种,横纹绿虎天牛Chlorophorus copiosus Holzschuh;艳虎天牛属Rhaphuma Pascoe中国2新纪录种,回纹艳虎天牛Rhaphuma lanzhui Holzschuh和箭纹艳虎天牛Rhaphuma illicata Holzschuh;刺虎天牛属Demonax T homson中国3新纪录种,断纹刺虎天牛Demonax traudae Holzschuh,蔷薇刺虎天牛Demonax rosae Holzschuh和格氏刺虎天牛Demonax gertrudae Holzschuh。研究标本保存在西南大学昆虫标本馆。     

天牛亚科中国四新纪录种(鞘翅目,天牛科)

记述了天牛亚科虎天牛族4新纪录种,刺虎天牛属Demonax Thomson的八点刺虎天牛D.contrarius Holzschuh;艳虎天牛属Rhaphuma Pascoe的独艳虎天牛R.unigena Holzschuh;绿虎天牛属Chlorophorus Chevrolat的胖绿虎天牛C.proannulatus Gressitt & Rondon;瘦棍腿天牛属Stenodryas Bates的四纹瘦棍腿天牛S.nigromaculatus(Gardner).研究标本保存在西南大学昆虫标本馆.     

SIX NEW RECORD SPECIES OF CLYTINI FROM CHINA ( COLEOPTER,CERAMBYCIDAE, CERAMBYCINAE)

共记述了绿虎天牛属Chlorophorus Chevrolat中国1新纪录种,横纹绿虎天牛Chlorophorus copiosus Holzschuh;艳虎天牛属Rhaphuma Pascoe中国2新纪录种,回纹艳虎天生Rhaphuma lanzhui Holzschuh和箭纹艳虎天牛Rhaphuma illicata Holzschuh;刺虎大牛属Demnonax Thomson中国3新纪录种,断纹刺虎天牛Demonax traudae Holzschuh,蔷薇刺虎天牛Demonax rosae Holzschuh和格氏刺虎天牛Demnonax gertrudae Holzschuh.研究标本保存存西南大学昆虫标本馆.     

我国七种绿虎天牛属幼虫(鞘翅目:天牛科)

绿虎天牛属Chlorophorus属于天牛亚科Cerambycinae,虎天牛族Clytini,包括许多常见害虫。本文记载了该属七种幼虫形态,它们是裂纹绿虎天牛Ch. separatus Gressitt、槐绿虎天牛Ch. diadema (Motschulsky)、澳门绿虎天牛Ch. macaumensis (Chevrolat)、半环绿虎天牛Ch. reductus Pic、刺槐绿虎天牛Ch. sulcaticeps Pic、榄绿虎天牛Ch. eleodes (Fairmaire)和竹绿虎天牛 Ch. annularis(Fairmaire)。竹绿虎天牛和刺槐绿虎天牛在浙江、福建和广东各地严重为害竹林,澳门绿虎天牛在海南岛为害咖啡树     

北京的脊虎天牛属分类研究(鞘翅目:天牛科)

对分布于北京的10种脊虎天牛属甲虫开展了分类研究,恢复了北京脊虎天牛Xylotrechus pekingensis Pic,1939的地位,不再是宽带脊虎天牛Xylotrechus yanoi Gressitt,1934的异名,报道了本种在河北和陕西的新分布记录.提出双带脊虎天牛Xylotrechus bifenestratus Pic,1916是四带脊虎天牛Xylotrechus polyzonus (Fairmaire,1888)的新异名,两者的模式标本均产自北京.本文还报道了3种北京新记录种:显纹脊虎天牛X.ibex (Gebler,1825)、葡脊虎天牛X.pyrrhoderus Bates,1873和黑胸脊虎天牛X.robusticollis (Pic,1936).通过检视标本,很多新的分布信息被加入到相关的种类.最后本文提供了分布于北京的10种脊虎天牛属甲虫的分种检索表.     

思茅咖啡天牛种群构成与危害的时空特性研究

通过室内饲养和野外调查,本文对思茅市咖啡天牛种群的构成,发生时期危害的时空特性及幼虫蛀孔部位的选择性进行了研究。结果表明,危害思茅地区咖啡的天牛种群由旋皮天牛和灭字虎天牛构成 ,在不同时间两个种群的构成比例不同。施皮天牛是优势种心化高峰期为４月１８－３０日,灭字虎天牛成虫主要在１０－１２月出现;在咖啡树中天牛幼虫的频数分布具有明显特征,１头虫的分布频数最高;     

Genome-wide identification and analysis of genes encoding cuticular proteins in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

The multifunctional insect cuticle serves as the exoskeleton, determines body shape, restricts water loss, provides attachment sites for muscles and internal organs and is a formidable barrier to invaders. It is morphologically divided into three layers, including envelope, epicuticle, and procuticle and is composed of chitin and cuticular proteins (CPs). Annotation of CPs and their cognate genes may help understand the structure and functions of insect cuticles. In this paper, we interrogated the genome of Pteromalus puparum, an endoparasitoid wasp that parasitizes Pieris rapae and Papilio xuthus pupae, and identified 82 genes encoding CPs belonging to six CP families, including 62 in the CPR family, 8 in CPAP3, 5 in CPF/CPFL, 2 low complexity proteins, 2 in TWDL, and 3 in Apidermin. We used six RNA-seq libraries to determine CP gene expression profiles through development and compared the cuticle hydrophobicity between the P. puparum and the ectoparasitoid Nasonia vitripennis based on GRAVY values of CPR sequences. In the Nasonia-Pteromalus comparison, we found in both N. vitripennis and P. puparum, the peak of their CPR hydrophobicity displayed at their pupal stage, whereas their adult stage showed the lowest level. Except at the adult stage, the CPR hydrophobicity in N. vitripennis is always higher than P. puparum. Finally, we identified three novel Apidermin genes, a family found solely in Hymenoptera and revealed a new sequence feature of this family. This new information contributes to a broader understanding of insect CPs generally.     

The CPCFC cuticular protein family: Anatomical and cuticular locations in Anopheles gambiae and distribution throughout Pancrustacea

Arthropod cuticles have, in addition to chitin, many structural proteins belonging to diverse families. Information is sparse about how these different cuticular proteins contribute to the cuticle. Most cuticular proteins lack cysteine with the exception of two families (CPAP1 and CPAP3), recently described, and the one other that we now report on that has a motif of 16 amino acids first identified in a protein, Bc-NCP1, from the cuticle of nymphs of the cockroach, Blaberus craniifer (Jensen et al., 1997). This motif turns out to be present as two or three copies in one or two proteins in species from many orders of Hexapoda. We have named the family of cuticular proteins with this motif CPCFC, based on its unique feature of having two cysteines interrupted by five amino acids (C-X(5)-C). Analysis of the single member of the family in Anopheles gambiae (AgamCPCFC1) revealed that its mRNA is most abundant immediately following ecdysis in larvae, pupae and adults. The mRNA is localized primarily in epidermis that secretes hard cuticle, sclerites, setae, head capsules, appendages and spermatheca. EM immunolocalization revealed the presence of the protein, generally in endocuticle of legs and antennae. A phylogenetic analysis found proteins bearing this motif in 14 orders of Hexapoda, but not in some species for which there are complete genomic data. Proteins were much longer in Coleoptera and Diptera than in other orders. In contrast to the 1 and occasionally 2 copies in other species, a dragonfly, Ladona fulva, has at least 14 genes coding for family members. CPCFC proteins were present in four classes of Crustacea with 5 repeats in one species, and motifs that ended C-X(7)-C in Malacostraca. They were not detected, except as obvious contaminants, in any other arthropod subphyla or in any other phylum.The conservation of CPCFC proteins throughout the Pancrustacea and the small number of copies in individual species indicate that, when present, these proteins are serving important functions worthy of further study.     

Knockdown of specific cuticular proteins analogous to peritrophin 3 genes disrupt larval and ovarian development in Bactrocera dorsalis (Diptera: Tephritidae)

Cuticular proteins (CPs) are critical components of the insect cuticle and play important roles in maintaining normal insect development and defense against various environmental stresses. The oriental fruit fly (Bactrocera dorsalis) is one of the most destructive pests worldwide, and its eight CPs analogous to peritrophin 3 (BdCPAP3) family genes have been identified in our previous study. In the present study, we further explored the possible roles of CPAP3 genes in B. dorsalis development. Each sequence of BdCPAP3 genes contained three conserved ChtBD2 (chitin-binding) domains. Spatial and temporal expression patterns revealed that the four BdCPAP3 genes (BdCPAP3-A1, B, E, and E2) might play important roles in larval pupariation of B. dorsalis. Moreover, treatment with a juvenile hormone analog (methoprene) significantly restricted expression of these four CPAP3 genes, whereas treatment with 20-hydroxy-ecdysone induced expression. The RNA interference (RNAi) results revealed that down-regulated CPAP3 genes led to significant delay of pupariation, and injection of dsBdCPAP3-E into 5-d-old B. dorsalis larvae caused approximately 40% mortality. Interestingly, we also confirmed that BdCPAP3-D2 was involved in B. dorsalis ovarian development. This study showed that some specific CPAP3 genes had crucial roles in B. dorsalis development, and these CP genes could be used as potential targets to control this pest via RNAi.     

Characterization of RR-1 and RR-2 cuticular proteins from Myzus persicae

A cDNA library for Myzus persicae has served to identify sequences coding for cuticular proteins (CPs) with RR-1 and RR-2 consensus. Two putative CPs showed a common RR-2 chitin binding domain (CBD) but differed in their C and N terminals. Two other predicted CPs showed a typical RR-1 CBD but differed in size and sequence of the C and N terminals. An additional sequence encoding for a protein that showed terminal amino acid repeats similar to those of putative CPs from M. persicae, but lacked the R & R consensus, was also described. A comparison of the sequences obtained from the cDNA library with those attained from the genomic DNA, confirmed their identity as cuticular proteins genes. Presence of introns was revealed in the Mpcp4 and Mpcp5 genes coding for CPs with an RR-1 consensus. The Mpcp4 has a single large intron, while the Mpcp5 has two shorter ones. Introns were not found in the Mpcp2 and Mpcp3 genes encoding for CPs with RR-2 consensus. Differences were also noticed for 3' UTR and 5' UTR of both the RR-1 and RR-2 CPs. CPs genes were expressed in bacteria, and the resulting protein was identified as a CP by amino acid sequencing.     

A novel gene family in moss (Physcomitrella patens) shows sequence homology and a phylogenetic relationship with the TIR-NBS class of plant disease resistance genes

Plant disease resistance (R) genes encode proteins in which several motifs of the nucleotide-binding region (NBS) are highly conserved. Using degenerate primers designed according to the kinase 1 (P-loop) and hydrophobic (HD) motifs of the R gene NBS domains, homologous sequences were cloned from moss (Physcomitrella patens; phylum Bryophyta) representing an ancient nonvascular plant. A novel gene family (PpC) with at least eight homologous members was found. Expression of five members was detected. The level of expression was dependent on the developmental stage of moss, being higher in the gametophyte tissue than in the protonema tissue. The PpCs contained the conserved motifs characteristic of the NBS regions of R genes, and a kinase domain was found upstream from the NBS region. Phylogenetic analysis using the deduced NBS amino acid sequences of the PpCs and the plant genes available in databanks indicated that the PpCs show the closest relationship with the TIR-NBS class of R genes. No significant similarity to plant genes other than R genes was observed. These findings shed novel light on the evolutionary history of the R gene families, suggesting that the NBS region characteristic of the TIR-NBS class of R-like genes evolved prior to the evolutionary differentiation of vascular and nonvascular plants.     

Divergent signature motifs of nucleotide binding domains of ABC multidrug transporter, CaCdr1p of pathogenic Candida albicans, are functionally asymmetric and noninterchangeable

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.     

Distribution of cuticular proteins in different structures of adult Anopheles gambiae

Anopheles gambiae devotes over 2% (295) of its protein coding genes to structural cuticular proteins (CPs) that have been classified into 13 different families plus ten low complexity proteins not assigned to families. Small groups of genes code for identical proteins reducing the total number of unique cuticular proteins to 282. Is the large number because different structures utilize different CPs, or are all of the genes widely expressed? We used LC-MS/MS to learn how many products of these genes were found in five adult structures: Johnston's organs, the remainder of the male antennae, eye lenses, legs, and wings. Data were analyzed against both the entire proteome and a smaller database of just CPs. We recovered unique peptides for 97 CPs and shared peptides for another 35. Members of 11 of the 13 families were recovered as well as some unclassified. Only 11 CPs were present exclusively in only one structure while 43 CPs were recovered from all five structures. A quantitative analysis, using normalized spectral counts, revealed that only a few CPs were abundant in each structure. When the MS/MS data were run against the entire proteome, the majority of the top hits were to CPs, but peptides were recovered from an additional 467 proteins. CP peptides were frequently recovered from chitin-binding domains, confirming that protein-chitin interactions are not mediated by covalent bonds. Comparison with three other MS/MS analyses of cuticles or cuticle-rich structures augmented the current analysis. Our findings provide new insights into the composition of different mosquito structures and reveal the complexity of selection and utilization of genes coding for structural cuticular proteins.