柞蚕空胴病病原拮抗菌的分离、鉴定及防治效果研究

【目的】本研究从辽宁省蚕业科学研究所柞园土壤中分离筛选对柞蚕空胴病病原菌有显著拮抗作用的细菌,为该病的生物防治奠定研究基础。【方法】采用稀释涂布平板法分离柞园土壤中细菌,利用抑菌圈法筛选拮抗效果显著的菌株;根据形态学、生理生化及分子生物学对拮抗菌进行鉴定;利用自然转化法对拮抗菌进行荧光蛋白标记,测定其在柞树叶片和柞蚕肠道内的定殖规律,并对其室内和野外防效进行测定。【结果】从柞园土壤中分离获得87株细菌,其中BF-49对柞蚕肠球菌的拮抗效果显著(P<0.001);鉴定结果显示该BF-49与贝莱斯芽孢杆菌(Bacillus velezensis)的亲缘关系最近,故将该菌株鉴定为Bacillus velezensis,命名为B. velezensis BF-49。荧光蛋白标记菌株BF-49-GFP在柞蚕肠道中能定殖5d,在柞树叶片上接种20d后浓度仍达1.25×104CFU/g。BF-49发酵液的10倍稀释液对柞蚕空胴病的室内防效为78.25%,野外防效为74.42%,均显著高于对照药剂。【结论】筛选获得的B. velezensis BF-49对柞蚕空胴病防效显著,可作为开发柞蚕空胴病...     

Characterization and functional study of a Cecropin‐like peptide from the Chinese oak silkworm,Antheraea pernyi

In present study, a Cecropin‐like peptide from Antheraea pernyi (ApCec) was cloned and characterized. The full‐length ApCec cDNA encoded a protein with 64 amino acids including a putative 22‐amino‐acid signal peptide, a 4‐amino‐acid propeptide, and a 38‐amino‐acid mature peptide. ApCec gene was highly expressed in Malpighian tubules of A. pernyi after induction for 24 h by Escherichia coli in PBS. Pro‐ApCec (including propeptide and mature peptide) and M‐ApCec (just mature peptide) were synthesized chemically and analyzed by HPLC and mass spectroscopy. The antibacterial activity of M‐ApCec is more potent than pro‐ApCec against E. coli K12 or B. subtilus in both minimum inhibitory concentration and inhibition zone assays. Hemolytic assay results showed M‐ApCec possessed a low cytotoxicity to mammalian cells. The secondary structure of M‐ApCec forms α‐helical structure, shown by circular dichroism spectroscopy. Transmission electron microscopy analysis suggested that M‐ApCec killed bacteria by disrupting bacterial cell membrane integrity. Our results indicate ApCec may play an important role in defending from pathogenic bacteria in A. pernyi, and it may be as a potential candidate for applications in antibacterial drug development and agriculture.     

A chromosome‐scale genome assembly of Antheraea pernyi (Saturniidae,Lepidoptera)

Antheraea pernyi is a semi‐domesticated lepidopteran insect species valuable to the silk industry, human health, and ecological tourism. Owing to its economic influence and developmental properties, it serves as an ideal model for investigating divergence of the Bombycoidea super family. However, studies on the karyotype evolution and functional genomics of A. pernyi are limited by scarce genomic resource. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first high‐quality A. pernyi genome from a single male individual. The genome is 720.67 Mb long with 49 chromosomes and a 13.77‐Mb scaffold N50. Approximately 441.75 Mb, accounting for 60.74% of the genome, was identified as repeats. The genome comprises 21,431 protein‐coding genes, 85.22% of which were functionally annotated. Comparative genomics analysis suggested that A. pernyi diverged from its common ancestor with A. yamamai ~30.3 million years ago, and that chromosome fission contributed to the increased chromosome number. The genome assembled in this work will not only facilitate future research on A. pernyi and related species but also help to progress comparative genomics analyses in Lepidoptera.     

Interspecific hybrids of Antheraea roylei and A. pernyi — a cytogenetic reassessment

Summary A rare case of interspecific hybridization between the Indian oak feeding silkworm Antheraea roylei (n=31) and Chinese oak feeding silkworm A. pernyi (n=49) yielding fertile and vigorous offspring is reported. The F₁ and the backcross (A. roylei X A. pernyi X A. pernyi male individuals of the above cross and the F₂₃ and F₃₂ male offspring derived from an earlier cross between another race of A. roylei (n=30) and A. pernyi (n=49) were cytogenetically analysed in order to study their chromosome dynamics. The F₁ hybrids showed 18 trivalents and 13 bivalents in the first meiotic prophase and metaphase. The backcross individuals possessed either 9 trivalents and 31 bivalents or 49 bivalents, in Metaphase I cells. The F₂₃ and F₃₂ individuals were karyotypically alike and exhibited 49 bivalents. The following conclusions were drawn from the above observations: (a) in spite of allopatry and karyotypic divergence in number, a high degree of homology exists between the chromosomal complements of the two species; (b) A. pernyi possibly evolved from A. roylei, during the course of which 18 chromosomes of the latter underwent fission to give rise to the 36 chromosomes of the former. This is demonstrated by trivalent formation and pairing affinities in F₁ hybrids; (c) selection has favoured the elimination of large A. roylei chromosomes which participated in trivalent formation in successive generations of inbred hybrids to establish a stable Karyotype like that of A. pernyi.     

Population dynamics of Dremomys pernyi and Callosciurus erythraeus in protective and non-protective pine forests at different ages

Four pine forests (6–10, 11–15, 16–20, and 31–40 year-old) located in the Cangshan Mountain and Erhai Lake National Reserve and 7 pine forests (1–5, 6–10, 11–15, 16–20, 21–30, 31–40, and more than 50 year-old) located in the non-protective area near the national reserve were selected. Three replications of each forest was set and a total of 33 sites were investigated. At each site, we quantified 6 habitat variables (species richness, abundance, and percentage of grasses and shrubs coverage respectively at the bottom layer of forests) within randomly determined 5 m × 5 m areas. One hundred cages were set in five lines at each site to trap small mammals, whose species and numbers were recorded. Dominance of Dremomys pernyi and Callosciurus erythraeus in small mammal communities, time niche breadth, and time niche overlap between the two small mammals were calculated, respectively. Step-wise regression was used to analyze the relationship between small mammals and habitat factors. Our results indicated that D. pernyi occurred earlier than C. erythraeus in protective pine forests. D. pernyi was captured in 6–10 year-old forest initially, and C. erythraeus was captured in 16–20 year-old forest initially. D. pernyi and C. erythraeus were captured in the 31–40 and 21–30 year-old forests initially in the non-protective area, respectively. Populations of D. pernyi and C. erythraeus in the 31–40 year-old protective forests were 3 and 3.75 times of those in the same-aged non-protective forests, respectively. Shrubs significantly influenced the populations of the two small mammals. The population of D. pernyi was positively correlated with the density of shrubs; the population of C. allosciurus erythraeus was positively correlated with the coverage of shrubs, and negatively correlated with the coverage of grasses. D. remomys pernyi and C. allosciurus erythraeus were important for pine forests to scatter pine seeds. Human activities in the nonprotective pine forests decreased the vegetation heterogeneity at the bottom layer of pine forests, postponed the occurrence of D. pernyi and C. erythraeus, and decreased the populations of the two small mammals. __________ Translated from Zoological Research, 2006, 27(1): 29–33 [译自: 动物学 研究]     

Analysis of oral infection and helicase gene of the nucleopolyhedroviruses isolated from Philosamia cynthia ricini and Antheraea pernyi

Philosamia cynthia ricini is an important commercial silkworm in Asia. In this report, a nucleopolyhedrovirus isolated from P. cynthia ricini (PhcyNPV) larva was purified and compared with Antheraea pernyi nucleopolyhedrovirus (AnpeNPV), a pathogen of A. pernyi, another commercial silkworm in China. The two viruses had similar polyhedral morphology and shared high sequence homologue of viral fragments including the p143 gene. However, the restriction fragments, digested with SalI, XhoI, HindIII and PstI, respectively, were different. The cross-infectivity of the two viruses was also tested. AnpeNPV caused 57% mortality in larvae of P. cynthia rici, whereas PcrNPV did not kill larvae of A. pernyi. Results indicated that PhcyNPV and AnpeNPV had closed relatedness, and that PhcyNPV might be a variant of AnpeNPV.     

Simplicillium chinense: a biological control agent against plant parasitic nematodes

Simplicillium chinense (Snef5) was isolated from Antheraea pernyi pupae and identified by morphological and molecular methods. Culture filtrates of Snef5 showed a high level of nematicidal activity. It is the first time to report that S. chinense can be used as a biological control agent against plant parasitic nematodes.     

A novel hexamerin with an unexpected contribution to the prophenoloxidase activation system of the Chinese oak silkworm,Antheraea pernyi

Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap-hexamerin), which shows high homology with reported insect methionine-rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap-hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap-hexamerin to immune responses. In further immune functional studies, Ap-hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen-associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap-hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein.     

Epoxide hydrolase in the bulb mite Rhizoglyphus robini: properties and induction

Using styrene oxide as substrate, most of the epoxide hydrolase (EH) activity monitored in the bulb mite Rhizoglyphus robini was associated with the microsomal compartment. The microsomal and cytosolic EHs did not display any significant preference in hydrating trans stilbene oxide (TSO) and cis stilbene oxide (CSO). The microsomal EH, which has a Km value of 5×10^-5M and pH optimum of 7.8, was sensitive to ethanol and its activity was inhibited to a moderate extent by 4-fluorochalcone oxide, TSO, CSO and trans-chalcone oxide at a level of 10^-4M. Microsomal EH was considerably induced (4–5-fold) in mites feeding garlic and onion, or ingesting TSO-impregnated filter papers. Other epoxides like CSO, 2,4-dichlorostilbene oxide, methyl chalcone oxide and heptachlor epoxide displayed moderate induction levels (1.4–2.6-fold). Of the toxicants assayed only sodium phenobarbital was a potent inducer. Lindane, malathion and DDT did not stimulate EH activity and 3-methyl-cholanthrene was even inhibitory. A decrease in EH activity was observed with a number of phytochemicals tested such as sinigrin, flavone, menthol, trans--carotene, chalcone, allyl sulphide and trans-cinnamic acid.     

Antheraea pernyi silk fibroin maintains the immunosupressive properties of human bone marrow mesenchymal stem cells

We reported previously that regenerated Antheraea pernyi silk fibroin (A. pernyi SF) could support the attachment and growth of human bone marrow mesenchymal stem cells (hBMSCs). In this work, the immunosupressive effects of hBMSCs cultured on the A. pernyi SF films on T-cells were investigated in vitro. The production of IL-6, CD80, CD86 and HLA-DR by the hBMSCs was also observed. The study showed that hBMSCs cultured on the regenerated A. pernyi SF films still kept their immunosupression on T-cell proliferation and IL-2 secretion. Moreover, regenerated A. pernyi SF like regenerated Bombyx mori SF and collagen did not elicit T-cell proliferation but it could support the expression of IL-6 and surface antigen of hBMSCs. Regenerated A. pernyi SF can maintain the function of hBMSCs in immunomodulation and cytokines production, which has the potential utility of hBMSCs combined with A. pernyi SF in tissue replacement and repair.     

Effect of an exponential feeding regime on the production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Aims: To evaluate the effect of and exponential feeding regime on the production of epoxide hydrolase (EH) enzyme in recombinant Yarrowia lipolytica in comparison to a constant feed strategy. Methods and Results: An exponential feed model was developed and fermentations were fed at six different exponential rates. A twofold increase in EH productivity and a 15% increase in volumetric EH activity was obtained by applying exponential glucose feed rates in fed‐batch cultivation. These responses were modelled to obtain a theoretical optimum feed rate that was validated in duplicate fermentations. The model optimum of 0·06 h^?1 resulted in a volumetric EH activity of c. 5500 U l^?1 h^?1 and a maximum activity of 206 000 U l^?1. This correlated well with model predictions, with a variance of <10%. Conclusions: The use of an exponential feed strategy at a rate of 0·06 h ^{? 1} yielded best results for all key responses which show a clear improvement over a constant feed strategy. Significance and Impact of the Study: The study was the first evaluation of an exponential feed strategy on recombinant Y. lipolytica for the production of EH enzyme. The results suggest a strategy for the commercial production of a valuable pharmaceutical enzyme.     

Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in ~ 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of ~ 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.     

The reproductive capability of Ooencyrtus kuvanae reared on eggs of the factitious host Antheraea pernyi

Ooencyrtus kuvanae is a key egg parasitoid of Lymantria dispar (L.), an important defoliating pest in China and North America. We have developed mass rearing techniques for O. kuvanae and in this study compared the reproductive capacity of O. kuvanae when it was previously reared on the natural host, L. dispar or the factitious host, Antheraea pernyi Guerin‐Meneville. There was no significant difference in the oviposition period or total number of eggs laid between L. dispar‐reared and A. pernyi‐reared females. However, the mean number of offspring successfully emerging from those eggs was significantly larger from the A. pernyi‐reared females compared with L. dispar‐reared females. From this, we can conclude that, with respect to reproductive capability, eggs of A. pernyi are suitable factitious hosts for mass rearing of O. kuvanae.     

Functional expression and magnetic nanoparticle-based Immobilization of a protein-engineered marine fish epoxide hydrolase of <Emphasis Type="Italic">Mugil cephalus</Emphasis> for enantioselective hydrolysis of racemic styrene oxide

A triple-point mutated fish microsomal epoxide hydrolase (mEH) gene from Mugil cephalus was expressed in Escherichia coli in the presence of various chaperones to prevent protein aggregations. The enantioselective hydrolytic activity was more than doubled by co-expressing the EH mutant gene with pGro7 plasmid. The highly active EH mutant with a his-tag was immobilized onto magnetic silica assembled with NiO nanoparticles. The immobilized mEH mutant was re-used more than 10 times with less than 10% activity loss. (S)-Styrene oxide with 98% enantiopurity was repeatedly obtained with over 50% of the theoretical yield by the magnetically separable high-performance mEH mutant.     

Comparative homology modeling-inspired protein engineering for improvement of catalytic activity of Mugil cephalus epoxide hydrolase

The epoxide hydrolase (EH) of a marine fish, Mugil cephalus, was engineered to improve the catalytic activity based on comparative homology modeling. The 3-D crystal structure of the EH from Aspergillus niger was used as a template. A triple point mutant, F193Y for spatial orientation of the nucleophile (D199), W200L for removing electron density overlap between W200 and Y348, and E378D for good charge relay in the active site, was developed. The initial hydrolysis rate, the reaction time to reach 98 %ee, and yield were enhanced up to 35-fold, 26-fold and 32%, respectively, by homology modeling-inspired site-directed mutagenesis of M. cephalus EH.     

Pheromone receptors in Bombyx mori and Antheraea pernyi

Summary The cellular organization of freeze-substituted antennal sensilla trichodea, which contain the sex pheromone receptors, was studied in male silkmoths of two species (Bombyx mori, Bombycidae; Antheraea pernyi, Saturniidae). The cellular architecture of these sensilla is complex, but very similar in both species. A three-dimensional reconstruction of a sensillum trichodeum of B. mori is presented. Two receptor cells (in A. pernyi 1–3) and three auxiliary cells are present. Of the latter, only the thecogen cell forms a true sheath around the receptor cells. A unique thecogen-receptor cell junction extends over the entire area of contact. Septate junctions occur between all sensillar cells apically, and in the region of the axonal origin basally. Gap junctions are also found between all cells except the receptor cells. The trichogen and tormogen cells show many structural indications of secretory activity and are thought to secrete the receptor lymph. Their apical membrane bordering the receptor-lymph space is enlarged by microvilli and microlamellae, but only those of the trichogen cell show regularly arranged membrane particles (portasomes), indicating secretory specialization among the auxiliary cells. Epidermal cells are found as slender pillars between sensilla, but extend apically along the non-sensillar cuticle and basally along the basal lamina.     

繁殖寄主对赤眼蜂羧酸酯酶和乙酰胆碱酯酶的影响

通过测定赤眼蜂Trichogramma羧酸酯酶和乙酰胆碱酯酶的活性、对底物的亲和力以及对抑制剂的敏感度研究了繁殖寄主对松毛虫赤眼蜂T.dendrolimi和螟黄赤眼蜂T.chilonis的影响。柞蚕卵和米蛾卵繁殖的赤眼蜂羧酸酯酶对底物的亲和力有不同程度的影响,柞蚕卵繁殖的赤眼蜂羧酸酯酶对α-乙酸萘酯或β-乙酸萘酯的亲和力最高是米蛾卵的2倍以上。繁殖寄主对乙酰胆碱酯酶对底物亲和力没有明显的影响。米蛾卵繁殖的松毛虫赤眼蜂羧酸酯酶活性明显高于柞蚕卵繁殖的种群,而米蛾卵繁殖的螟黄赤眼蜂种群羧酸酯酶的活性明显低于柞蚕卵繁殖的种群。用柞蚕卵繁殖的松毛虫赤眼蜂种群对对氧磷的敏感度明显低于米蛾卵繁殖的种群,而增效磷则正好相反。繁殖寄主对松毛虫赤眼蜂吉林种群乙酰胆碱酯酶对DDVP和毒扁豆碱的敏感度没有明显的影响,而在松毛虫赤眼蜂广东种群和螟黄赤眼蜂中,柞蚕卵繁殖的种群乙酰胆碱酯酶对DDVP和毒扁豆碱的敏感度明显低于米蛾卵繁殖的种群。     

D2‐like dopamine receptors mediate regulation of pupal diapause in Chinese oak silkmoth Antheraea pernyi

The present study investigated the pharmacological properties of dopamine receptors that functioned in the termination of pupal diapause in the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). Dopamine receptors are classified according to their structure and function into two subfamilies as D1‐ and D2‐like receptors. D1‐like receptors activate, whereas D2‐like receptors inhibit, adenylate cyclase. We examined the effects of agonists and antagonists selective for D1‐ and D2‐like receptors on the diapause state. As A. pernyi is a long‐day species, pupal diapause is maintained during short days and can be terminated by exposure to a long‐day photoperiod. The D2‐like receptor‐selective agonist quinpirole delayed the timing of adult emergence under long days, and the D2‐receptor‐selective antagonist sulpiride terminated pupal diapause even under a short‐day photoperiod. The D1‐like receptor‐selective agonist and antagonist, SKF‐38393 and SCH‐23390, respectively, caused no significant effects on diapause pupae. These results suggest that not D1‐ but D2‐like receptors mediated diapause regulation in A. pernyi. This dopamine pathway appeared to block the termination of pupal diapause. Furthermore, the actions of the cAMP analog 8‐CPT‐cAMP and dopamine receptor antagonists upon diapause pupae were similar, which supports the notion that D2‐like receptors involved in diapause of this insect prevent adenylate cyclase from producing cAMP like vertebrate D2‐like receptors. Taken together, our findings suggest that dopamine blocked diapause termination through D2‐like receptors that inhibited adenylate cyclase in A. pernyi. During short days under which diapause was maintained in pupae, the dopaminergic mechanism might be stimulated to suppress cAMP levels in cells regulating diapause.     

Influence of pH on the expression of a recombinant epoxide hydrolase in Aspergillus niger

The filamentous fungus Aspergillus niger was investigated in relation to its ability to produce a soluble epoxide hydrolase (EH) (E.C. 3.3.2.3) belonging to the microsomal EH family. This EH is a highly useful biocatalyst for kinetic resolution of racemic epoxides to give enantiopure building blocks. The production of EH on an industrial scale is still a major challenge and is linked to various optimization processes. In this work, production of protein and organic acids as a function of pH and cultivation time was investigated. The production of EH was highest (1000 U/L for p-nitrostyrene oxide) under acidic fermentation conditions (pH value of about 3). The metabolic flux toward production of organic acids and thereby acidification of the environment increased with an increasing pH value. At pH 7, nearly 50% of total carbon of the substrate was incorporated into organic acids, mainly gluconic and oxalic acid. Finally, the addition of protease inhibitors, antioxidants and cryoprotectants was investigated in relation to the stability of the EH during the downstream process. The determination of the pH dependence during fermentation and understanding of the parameters influencing the stability of the enzyme has allowed us to optimize intracellular expression. The EH has been easily isolated from the biomass with high activity (1.67 U/mg lyophilisate) in a robust process.