中国西部麦红吸浆虫种群遗传结构的RAPD分析(双翅目:瘿蚊科)

对采自我国西北春麦区武威市 ( 38°N,1 0 3°E)、皋兰县 ( 36.5°N,1 0 4°E)及冬麦区长安县 ( 34.2°N,1 0 9°E)的麦红吸浆虫 Sitodiplosis mosellana ( Gehin)种群和皋兰县的麦黄吸浆虫 Contarinia tritici( Kirby)种群用 4种引物 S1 8、S61、S50、S83进行了RAPD测定分析。结果表明 ,引物共产生 1 0 6个 RAPD标记 ,没有任何一个引物能单独区分所有个体 ,但四种引物扩增结果联合则可。引物在麦红吸浆虫的 3个地理种群之间没有产生各自的特征带谱 ,但在 2个种之间可扩增出鉴别图谱。聚类分析显示 :同种同地理种群的个体间遗传相似程度高 ,变异范围小 ;同种不同地理种群间的遗传相似程度变异范围大 ,部分与种群内个体间的遗传相似程度重叠 ;不同种之间的遗传相似程度最低。在麦红吸浆虫种群间 ,长安与皋兰的种群遗传关系最近 ,而与武威种群较远。以近缘属种麦黄吸浆虫作参照分析表明 ,麦红吸浆虫不同地理种群之间表现为种下的遗传差异。     

中国麦红吸浆虫(双翅目：瘿蚊科)不同地理种群的遗传结构

采用RAPD方法测定分析了我国麦红吸浆虫Sitodiplosis mosellana (Gehin) 10个地理种群的遗传结构。结果表明：5种引物在10个种群中共产生了326个RAPD标记,各地理种群之间没有产生各自的特征标记;不同种群间的遗传相似程度与其地理间距呈反比;种群内的遗传相似程度比种群间的高;利用UPGMA法可将其聚为冬麦区和春麦区两大类群。     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.     

Volatiles associated with different flower stages and leaves of Acacia cyclops and their potential role as host attractants for Dasineura dielsi (Diptera: Cecidomyiidae)

Acacia cyclops (Fabaceae) is an Australian species which was introduced into South Africa in the nineteenth century. Because of its invasive status in South Africa, a gall midge, Dasineura dielsi (Diptera: Cecidomyiidae), was released in 2001 in order to impact its reproduction by inducing galls on the flowers and thereby preventing seed set. Nothing is known about the cues used by D. dielsi for locating its host flowers. As part of an initial investigation into whether or not chemical cues might play a role in host finding, we analysed headspace samples of Acacia cyclops volatiles from leaves and reproductive parts at different stages (early bud, late bud, early flowering, and senescing flowering stages) using gas chromatography–mass spectrometry (GC–MS). In total, 72 different compounds were detected of which 62 were identified. The analyses showed that open flowers, the stage used by D. dielsi for oviposition, and yellow buds had similar odour compositions with (Z)-3-hexen-1-ol acetate, 4-oxoisophorone, (Z)-β-ocimene, an unknown aliphatic compound, heptadecane, and nonadecane dominating in open flowers. Leaf volatiles were distinct from those in the reproductive plant parts by their high relative amount of (Z)-β-ocimene. (Z)-3-Hexen-1-ol acetate had its maximum relative amount in the green bud samples and was much lower in the later floral stages. In contrast, 4-oxoisophorone peaked in yellow buds and open flowers with little or none of it found in younger or older stages. The volatile compounds of the different flower stages and leaves are discussed in relation to their potential role as attractants used by the biocontrol agent D. dielsi to locate its host plant.     

麦红吸浆虫唾腺EST-SSRs的信息分析及分子标记筛选

昆虫EST资源库的扩充为开发新的分子标记提供了宝贵的资源。本研究对NCBI的EST数据库中来源于麦红吸浆虫Sitodiplosis mosellana唾腺的1 217条EST序列进行了unigene组装、 SSR信息分析和EST-SSR分子标记筛选。结果表明： 在1 047个unigenes中共找到141个SSR位点, 分布于106个（10.12%）unigenes中, 平均每3.49 kb出现一个SSR位点。在1~6碱基重复基元中, 1~3碱基是主要重复类型, 占总SSR的97.16%以上。A/T（31.21%）, AC/GT（15.60%）和AAC/GTT（9.22%）分别是单、 双和三碱基中占优势的重复基元类型。利用Primer Premier 5.0软件对查找的EST SSRs进行引物设计, 并以麦红吸浆虫基因组DNA为模板, 对从中选出的26对SSR引物进行多态性检测。结果有20对（76.92%）引物能扩增出清晰的目的条带, 并且其中9对（45%）引物表现出多态性。多态性分析结果表明, 从9对EST-SSR引物中, 共检测到51个等位基因, 平均每个位点含有等位基因数为5.67, 平均期望杂合度为0.65, 平均多态信息含量为0.60。本研究能够为今后麦红吸浆虫的种群遗传结构与遗传多样性研究提供帮助。     

麦红吸浆虫不同滞育期四种糖代谢酶活力分析

海藻糖是麦红吸浆虫Sitodiplosis mosellana (Gehin)滞育期间储藏能量的主要物质,为弄清其在滞育期的积累机理,本文测定了麦红吸浆虫滞育前后和滞育期糖原磷酸化酶（Gpase）、己糖激酶（HK）、磷酸果糖激酶（PFK）和醛缩酶（ALD）这4种糖代谢酶活力的变化。结果表明: 麦红吸浆虫滞育前后这些糖代谢酶活力明显不同,滞育后Gpase 活力显著提高,糖酵解有关酶HK,PFK和ALD活力降低;滞育解除后,Gpase 活力降低,HK,PFK和ALD活力升高。滞育期间,4种糖代谢酶的活力均与滞育发育有关;同时Gpase 和PFK的活力也与环境温度有关,即夏、冬季高于春、秋季;同期不同滞育状态幼虫比较,裸露幼虫HK,PFK和ALD活力总是略高于结茧幼虫,Gpase则相反。滞育当年与第2年同期幼虫4种糖代谢酶的活力无显著差异。     

中国麦红吸浆虫Sitodiplosis mosellana (Gehin)地理种群mtDNA COⅡ序列的遗传变异

对麦红吸浆虫Sitodiplosis mosellana(Gehin)11个地理种群线粒体DNA(mtDNA)细胞色素C氧化酶亚基Ⅱ(COⅡ)的部分片段进行了测序,分析了核苷酸的组成,构建了NJ分子系统树。结果表明,在用于分析的447bp序列中,A T平均含量为78．6％,其中7个位点存在变异。NJ树显示,麦红吸浆虫各种群间的亲缘关系与地理分布关系密切：11个地理种群可明显地聚为两大族群,所有来自春麦区的种群(NH、QX、GT、GW、GG)和冬麦区(陕西长安SC)种群聚为族群Ⅰ,其它冬麦区种群(HT、HL、HX、AS、AF)聚为族群Ⅱ;陕西长安种群(SC)虽处于冬麦区,但与春麦区种群具有很高的遗传相似性。该结果支持将我国麦红吸浆虫的发生区域分为三个,即春麦发生区、冬春麦发生区、冬麦发生区。各地理种群间的遗传距离为0～0．016,表现为种下的遗传变异,这与RAPD分析得出的结果相一致。     

Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee

The incidence of nosemosis has increased in recent years due to an emerging infestation of Nosema ceranae in managed honey bee populations in much of the world. A real-time PCR assay was developed to facilitate detection and quantification of both Nosema apis and N. ceranae in both single bee and pooled samples. The assay is a multiplexed reaction in which both species are detected and quantified in a single reaction. The assay is highly sensitive and can detect single copies of the target sequence. Real-time PCR results were calibrated to spore counts generated by standard microscopy procedures. The assay was used to assess bees from commercial apiaries sampled in November 2008 and March 2009. Bees from each colony were pooled. A large amount of variation among colonies was evident, signifying the need to examine large numbers of colonies. Due to sampling constraints, a subset of colonies (from five apiaries) was sampled in both seasons. In November, N. apis levels were 1212 ± 148 spores/bee and N. ceranae levels were 51,073 ± 31,155 spores/bee. In March, no N. apis was detected, N. ceranae levels were 11,824 ± 6304 spores/bee. Changes in N. ceranae levels were evident among apiaries, some increasing and other decreasing. This demonstrates the need for thorough sampling of apiaries and the need for a rapid test for both detection and quantification of both Nosema spp. This assay provides the opportunity for detailed study of disease resistance, infection kinetics, and improvement of disease management practices for honey bees.     

Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules

Apis mellifera (Amel) relies on its olfactory system to detect and identify new-sources of floral food. The Odorant-Binding Proteins (OBPs) are the first proteins involved in odorant recognition and interaction, before activation of the olfactory receptors. The Amel genome possess a set of 21 OBPs, much fewer compared to the 60-70 OBPs found in Diptera genomes. We have undertaken a structural proteomics study of Amel OBPs, alone or in complex with odorant or model compounds. We report here the first 3D structure of a member of the C-minus class OBPs, AmelOBP14, characterized by only two disulfide bridges of the three typical of classical OBPs. We show that AmelOBP14 possesses a core of 6 α-helices comparable to that of classical OBPs, and an extra exposed C-terminal helix. Its binding site is located within this core and is completely closed. Fluorescent experiments using 1-NPN displacement demonstrate that AmelOBP14 is able to bind several compounds with sub micromolar dissociation constants, among which citralva and eugenol exhibit the highest affinities. We have determined the structures of AmelOBP14 in complex with 1-NPN, eugenol and citralva, explaining their strong binding. Finally, by introducing a double cysteine mutant at positions 44 and 97, we show that a third disulfide bridge was formed in the same position as in classical OBPs without disturbing the fold of AmelOBP14.     

Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea

Mago nashi (Mago) and Y14 proteins, highly conserved among eukaryotes, participate in mRNA localization and splicing, and as such play important roles in oogenesis, embryogenesis and germ-line sex determination during animal development. Here we identified mago (Acmago) and Y14 (AcY14) homologues derived from Antrodia cinnamomea. Acmago encodes 149 amino acids and AcY14 encodes 168 amino acids. Multiple amino acid sequence alignment as well as secondary and tertiary structure prediction showed that AcMago and AcY14 have similar protein structure to the reported crystal structures of other Mago and Y14 proteins. During fungal development both Acmago and AcY14 genes were abundantly expressed in natural basidiomes. This is the first report of the molecular characterization and expression analysis of the mago and Y14 genes from fungi.     

Morphological diversity of first instar larvae in Miltogramma subgenus Pediasiomyia (Diptera: Sarcophagidae, Miltogramminae)

The morphology of first instar larvae of three species of Miltogramma Meigen subgenus Pediasiomyia Rohdendorf is described using SEM and light microscope techniques. Miltogramma chrysochlamys (Rohdendorf), Miltogramma margiana (Rohdendorf) and Miltogramma przhevalskyi (Rohdendorf) share with other satellite flies the presence of an elongated sensillum basiconicum of the maxillary palpus and numerous longitudinal cuticular ridges on the integument of all segments; and they share with other Miltogramma spp. a maxillary palpus situated on a more or less raised base. While the first instar M. margiana is very similar to first instars of Miltogramma (sensu stricto), first instars of M. chrysochlamys and M. przhevalskyi share several features unique among Miltogramminae: antennal dome situated on a flat or indistinct basal ring, antennal dome flattened, border between pseudocephalon and first thoracic segment with a pair of fleshy processes, and basal ring with trichoid sensillum. First instars of M. chrysochlamys and M. przhevalskyi are unique in having both sb1 and sb2 of the sensilla basiconica of the maxillary palpus elongated, and M. przhevalskyi has an unpaired, median proleg on most abdominal segments.     

Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development

It has been extensively documented that exposure of amphibians and teleost fish to exogenous steroid hormones like estrogen, androgen, xenoestrogen or steroid biosynthesis inhibitors can impair their gonadal development or induce sex reversal against genotypic sex. However, the molecular pathways underlying sexual development and the effects of sex steroids or other exogenous hormones in these aquatic vertebrates remain elusive. Recently, a germ plasm-associated piRNA (piwi-interacting RNA) pathway has been shown to be a determinant in the development of animal gonadal germline cells. In the current study, we examined whether this piRNA pathway is involved in the regulation of sex steroid hormones in gonadal development. We firstly established developmental expression patterns of three key piRNA pathway genes (mael, piwi and vasa), during Silurana (Xenopus) tropicalis embryogenesis and early larval development. All three genes exhibit high expression at early developmental stages and have significantly decreased expression thereafter, indicating a very active involvement of piRNA pathway at the beginning of embryogenesis. We further examined gene expression changes of those genes in frog larvae exposed to two sex steroid biosynthesis inhibitors, fadrozole and finasteride, both of which are known to result in male-biased or female-biased phenotypes, respectively. We found that fadrozole and finasteride exposures increased the expression of piRNA pathway genes such as mael and vasa at the larval stage when the expression of piRNA pathway genes is programmed to be very low. Therefore, our results indicate that the piRNA pathway is likely a common pathway by which different sex steroid hormones regulate gonadal sex differentiation.