Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm / 生物工程学报

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

SYNERGISTIC EFFECT OF HONEY AND LEMON JUICE-ENRICHED MULBERRY DIETS ON THE GROWTH OF SILKWORM, BOMBYX MORI DURING METAMORPHOSIS

Synergistic impact of honey and lemon juice-enriched mulberry diets on the growth of Bombyx mori was studied. The body size progressively increased during larval stage, but declined during pupal and adult stages. The nutrient diets promoted larval growth and positively modulated pupal and adult growth. The larval growth curves are typical Gompertz trajectories that re?ected the growth promoting nature of honey and lemon juice. The log-based growth curves were used to derive critical larval body size determinants that control molting and metamorphosis. The nutrient diets improved critical body size determinants without affecting their time schedules. The compound periodical growth rates showed instar-speci?c and stage-speci?c variations. The size speci?c growth rates in body mass, length and perimeter dimensions indicated the prevalence of an effective mass management mechanism as de?ned in the Hutchinson's investment principle. The silkworm recorded either higher or lower growth ratios indicating deviation from the Dyar's constancy rule.

Antibacterial activity of Bombyx mori and Samia cynthia ricini sericin proteins against Escherichia coli and Staphylococcus aureus

Aims@#This study was designed to investigate the antibacterial properties of the sericin protein Bombyx mori and Samia cynthia ricini against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus strains.@*Methodology and results@#Sericin protein was extracted from the cocoons of B. mori races A, B, C, D, F1-1, F1-2 and S. ricini from Indonesia using a degumming process. The extracted sericin protein was evaluated as anti-microbials using Kirby Breuer disc diffusion, spectrometer measurement in decreasing OD value and imaging scanning electron microscope (SEM). The tests revealed that sericin protein from B. mori was more capable of inhibiting bacteria S. aureus than E. coli. Sericin protein from S. ricini was also capable of strongly inhibiting both S. aureus and E. coli bacteria. The sericin proteins of B. mori and S. ricini were found to reduce the number of S. aureus and E. coli bacteria as the OD value decreased at a concentration of 9%. The SEM imaging suggests that sericin B. mori and S. ricini proteins caused changes in cell morphology in S. aureus and E. coli bacteria, resulting in bacterial cell function disruption and death.@*Conclusion, significance and impact of study@#Bombyx mori and S. ricini sericin proteins were found to act against S. aureus and E. coli bacteria. Therefore, sericin protein has a greater antibacterial activity against Gram-positive strain S. aureus.

Resveratrol elongates the lifespan and improves antioxidant activity in the silkworm Bombyx mori / 药物分析学报

A number of research has shown that the plant polyphenol resveratrol,one of the most prominent small molecules,has beneficial protective effects in multiple organisms,including worms,flies,and killifish.To understand the effects of resveratrol on lifespan,we evaluated its effects in the silkworm Bombyx mori.In this study,we found that lifespan was significantly prolonged in both female and male silkworms treated with resveratrol.Silkworm larval weight was significantly increased from day 3 of the 5th larval instar(L5D3) to day 7 of the 5th larval instar (L5D7).However,the weight of the pupa,cocoon,and total cocoon was not significantly different in female silkworms with resveratrol treatment than that in controls.Meanwhile,resveratrol significantly improved the thermotolerance of the silkworms,which enhanced their survival rate.Moreover,antioxidant activity was increased by resveratrol in both female and male silkworms.Furthermore,an antioxidant-related signalling pathway,SIRT7-FoxO-GST,was activated in silkworms with resveratrol treatment.Collectively,these results help us to understand the molecular pathways underlying resveratrol induced pro-longevity effects and indicate that silkworm is a promising animal model for evaluating the effects of lifespan-extending drugs.

Development of a CRISPR/Cpf1 gene editing system in silkworm Bombyx mori / 生物工程学报

The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.

Study on HPLC Fingerprint of Bombyx mori from Different Producing Areas and Content Determination of Multi- component / 中国药房

OBJECTIVE：To establish the fingerprint of Bombyx mori and the method for the content determination of multi- components，and to provide reference for comprehensive quality evaluation of B. mori . METHODS ：Using 18 batches of B. mori from different producing areas as samples ，HPLC method was used. The column was Shiseido CAPCELL PAK C 18 AQ S 5 with mobile phase consisted of methanol- 0.05 mol/L potassium dihydrogen phosphate solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelength was 260 nm，and column temperature was set at 30 ℃. The sample size was 10 μL. HPLC fingerprint analysis and similarity evaluation were performed by using TCM Chromatogram Fingerprint Similarity Evaluation System（2012 edition），and the chromatographic peak was identified by comparing with the chromatogram of reference substance. The contents of 4 nucleosides as uracil ，guanine，xanthine，uridine were determined . RESULTS ：A total of 16 common peaks were identified in HPLC fingerprint for 18 batches of B. mori ，and peaks 3，6 ，7 and 8 were identified as uracil ，guanine，xanthine and uridine. The similarity of sample chromatogram with control fingerprint were 0.912-1.000. The linear range of uracil ，guanine， xanthine and uridine were 5.34-534，5.28-528，5.06-506，5.195-519.5 μg/mL（r≥0.999 8）. The limits of detection were 0.032 4， 0.032 0，0.030 7，0.031 2 μg/mL，and the limits of quantitation were 0.106 8，0.105 6，0.101 2，0.103 0 μg/mL. RSDs of precision，reproducibility and stability tests （24 h）were all lower than 1.00％（n＝6）. Average recoveries were 100.15％-102.95％， and RSD s were all lower than 2.00％（n＝9）. The content determination results showed that the content of uracil ，guanine， xanthine and uridine of B. mori from different producing areas were 0.41％-2.46％，0.37％-1.98％，0.72％-2.63％，0.94％-3.67％， respectively. CONCLUSIONS ：Established HPLC fingerprint and content determination method of 4 nucleosides were specific ， accurate and reliable ，which can be used for the quality evaluation and control of B. mori .

Sericin as treatment of obesity: morphophysiological effects in obese mice fed with high-fat diet / Sericina como tratamento da obesidade: efeitos morfofisiológicos de camundongos obesos por dieta hiperlipídica

ABSTRACT Objective To investigate the effects of sericin extracted from silkworm Bombyx mori cocoon on morphophysiological parameters in mice with obesity induced by high-fat diet. Methods Male C57Bl6 mice aged 9 weeks were allocated to one of two groups - Control and Obese, and fed a standard or high-fat diet for 10 weeks, respectively. Mice were then further subdivided into four groups with seven mice each, as follows: Control, Control-Sericin, Obese, and Obese-Sericin. The standard or high fat diet was given for 4 more weeks; sericin (1,000mg/kg body weight) was given orally to mice in the Control-Sericin and Obese-Sericin Groups during this period. Weight gain, food intake, fecal weight, fecal lipid content, gut motility and glucose tolerance were monitored. At the end of experimental period, plasma was collected for biochemical analysis. Samples of white adipose tissue, liver and jejunum were collected and processed for light microscopy analysis; liver fragments were used for lipid content determination. Results Obese mice experienced significantly greater weight gain and fat accumulation and had higher total cholesterol and glucose levels compared to controls. Retroperitoneal and periepididymal adipocyte hypertrophy, development of hepatic steatosis, increased cholesterol and triglyceride levels and morphometric changes in the jejunal wall were observed. Conclusion Physiological changes induced by obesity were not fully reverted by sericin; however, sericin treatment restored jejunal morphometry and increased lipid excretion in feces in obese mice, suggesting potential anti-obesity effects.

RESUMO Objetivo Investigar os efeitos da sericina extraída de casulos de Bombyx mori na morfofisiologia de camundongos com obesidade induzida por dieta hiperlipídica. Métodos Camundongos machos C57Bl6, com 9 semanas de idade, foram distribuídos em Grupos Controle e Obeso, que receberam ração padrão para roedores ou dieta hiperlipídica por 10 semanas, respectivamente. Posteriormente, os animais foram redistribuídos em quatro grupos, com sete animais cada: Controle, Controle-Sericina, Obeso e Obeso-Sericina. Os animais permaneceram recebendo ração padrão ou hiperlipídica por 4 semanas, período no qual a sericina foi administrada oralmente na dose de 1.000mg/kg de massa corporal aos Grupos Controle-Sericina e Obeso-Sericina. Parâmetros fisiológicos, como ganho de peso, consumo alimentar, peso das fezes em análise de lipídios fecais, motilidade intestinal e tolerância à glicose foram monitorados. Ao término do experimento, o plasma foi coletado para dosagens bioquímicas e fragmentos de tecido adiposo branco; fígado e jejuno foram processados para análises histológicas, e amostras hepáticas foram usadas para determinação lipídica. Resultados Camundongos obesos apresentaram ganho de peso e acúmulo de gordura significativamente maior que os controles, aumento do colesterol total e glicemia. Houve hipertrofia dos adipócitos retroperitoneais e periepididimais, instalação de esteatose e aumento do colesterol e triglicerídeos hepáticos, bem como alteração morfométrica da parede jejunal. Conclusão O tratamento com sericina não reverteu todas as alterações fisiológicas promovidas pela obesidade, mas restaurou a morfometria jejunal e aumentou a quantidade de lipídios eliminados nas fezes dos camundongos obesos, apresentando-se como potencial tratamento para a obesidade.

Mechanism analysis of Anti-BmNPV resistant strain NC99R / 生物工程学报

Bombyx mori is a lepidopteran insect with important economic value. Bombyx mori nucleopolyhedrovirus (BmNPV) causes huge economic loss to silkworm industry in China every year. The objective of this study is to determine the anti-BmNPV mechanism of Bombyx mori strain NC99R, and to provide a basis for understanding the molecular mechanism of the silkworm resistance strain. The normal control Dazao (DZ) strain and the NC99R resistant strain were fed with occlusion bodies (OB). The median lethal dose (LD50) analysis of the DZ and NC99R showed that the LD50 of DZ was 1.2×10⁵ OBs/larva, while NC99R was 1.8×10⁶ OBs/larva. The LD50 of the NC99R was about 15 times higher than the DZ. The mortality of DZ and NC99R were analyzed, which were fed with 1×10⁶ OBs/larva and injection with 1×10⁶ BVs/larva. The results showed that the death peak of DZ was concentrated in the 4th to 6th day. And the death peak of NC99R was concentrated in the 6th to 8th day, with a delay of 1-2 days compared with the control. The BmNPV DNA copy number showed that the BmNPV genome in DZ proliferated rapidly. The copy number of BmNPV DNA in NC99R were increased slowly after oral infection and body injection. HE staining showed that midgut tissue has no significant difference between DZ and NC99R in the early stage of oral infection. At 96 h p.i., the nucleus of DZ midgut became larger and shedding. The NC99R had enlarged nuclei, but the cells were still arranged neatly. Finally, the expression of virus genes in different periods were analyzed by RT-PCR. The results indicated that the immediate early gene ie-1 expression levels began to down-regulate after 24 h p.i.. The early, late, and extremely late genes were also down-regulated, and finally maintained at a lower expression level.

Desarrollo y caracterización de películas de fibroina de seda para reparación condral / Development and characterization of silk fibroin films for condral reparation

RESUMEN La fibroína de seda es una proteína que ha demostrado ser un biomaterial con gran potencial en medicina regenerativa, por sus características de biocompatibilidad y su amplia posibilidad de modificación estructural permite ser usada como andamio favoreciendo procesos de crecimiento, diferenciación celular y la regeneración del tejido afectado. En este estudio se utilizaron capullos de gusano de seda Bombyx mori L., para la fabricación de películas de fibroína, los capullos fueron desgomados utilizando Na2CO3 0,02M, la fibroína obtenida se disolvió con LiBr 9,3M, el cual fue eliminado mediante diálisis y finalmente la solución de fibroína fue concentrada mediante contradiálisis. La fibroína fue servida en cajas de poliestireno, secadas a 90°C/24 horas y esterilizadas con etanol al 70%. Células madre mesenquimales fueron sembradas sobre estas películas de fibroína e inducidas a diferenciación utilizando un medio condrogénico especifico. La diferenciación fue evaluada por triplicado a los 14 y 21 días mediante extracción de ARN total, síntesis de ADN copia y amplificación por PCR de un grupo de genes específicos de cartílago empleando cebadores específicos. Se fabricaron películas de fibroína estables y resistentes que permitieron el crecimiento y la multiplicación celular, así como la diferenciación condrogénica evidenciada por la expresión de genes condrogenicos, no se afectó la viabilidad ni el recuento celular, las células interactuaron con el andamio evidenciado por el área de tapizado formado sobre la superficie de la película de fibroína. Finalmente se concluye que la fibroína de seda es un biomaterial que puede servir de andamio potencial para la regeneración de lesiones articulares.

ABSTRACT Silk fibroin is a protein that has been shown to be a biomaterial with great potential in regenerative medicine, due to its biocompatibility characteristics and its wide possibility of structural modification can be used as scaffold, favoring growth processes, cell differentiation and the regeneration of affected tissue. Bombix mori L. silkworm cocoons were used to make fibroin films, the silk fibroin were degummed using 0.02M Na2CO3, the obtained fibroin was dissolved with 9.3M LiBr, which was eliminated by dialysis and finally the fibroin solution was concentrated to 17% by counterdialysis. The fibroin was served in polystyrene boxes, dried at 90°C/24 hours and sterilized with 70% ethanol. The mesenchymal stem cells were seeded on fibroin films and induced differentiation using a specific chondrogenic medium. Differentiation was assessed in triplicate at 14 and 21 days by total RNA extraction, DNA synthesis copy and PCR amplification of a group of cartilage-specific genes using specific primers. Stable and resistant fibroin films that allowed cell growth and multiplication were fabricated, as well as the chondrogenic differentiation evidenced by the expression of chondrogenic genes, the viability and the cell count were not affected, the cells interacted with the scaffolding evidenced by the area of upholstery formed on the surface of the fibroin film. Finally, it is concluded that silk fibroin is a biomaterial that can serve as a potential scaffold for the regeneration of joint injuries.

Comparison of chemical constituents of wild silkworm cocoon and domestic silkworm cocoon by UHPLC-MS technology / 生物工程学报

Identifying and comparing the chemical constituents of wild silkworm cocoon and silkworm cocoon is of great significance for understanding the domestication of silkworm. In this study, we used high temperature and high pressure and methanol-water system to extract cocoon chemical constituents. We used UHPLC-MS to identify and compare cocoon chemical constituents of wild silkworm and domestic silkworm Dazao and Haoyue strains. The cocoon metabolic fingerprints of wild silkworm and domestic silkworm Dazao and Haoyue strains were obtained by using the UHPLC-MS in the positive ion mode and negative ion mode. By annotation, we found that cocoon chemical compounds with high abundances contained amino acids, flavonoids, alkaloids, terpenes, organic acids, and lignans. PLS-DA showed that the cocoon components were significantly different among the wild silkworm and two domestic silkworm strains Dazao and Haoyue. Proline, leucine/isoleucine and phenylalanine showed significantly higher abundances in the cocoon of domestic silkworm Dazao strain than in those of wild silkworm and domestic silkworm Haoyue strain. The flavonoid secondary metabolites are abundant in the Dazao cocoon, including quercetin, isoquercetin, quercetin 3-O-sophoroside, quercetin-3-O-α-L-rhamnoside, quercetin-3-O- rutinoside, and kaempferol. The other secondary metabolites, alkaloids, terpenes and lignans, showed higher abundances in the wild silkworm cocoon than in the domestic silkworm cocoon, including neurine, candicine, pilocarpidine, artemisiifolin, eupassopin, and eudesobovatol. By exposing cocoons to UV light and observing the green fluorescence of flavonoids, we found that Dazao cocoon had the most flavonoids, and Haoyue cocoon had least flavonoids and wild silkworm cocoon had mediate flavonoids. Alkaloids and organic acids are good anti-insect and antimicrobial agents, which have high abundance in the wild silkworm cocoon and could enhance the defense ability of wild silkworm cocoon. Flavonoids are abundant in the cocoon of domestic silkworm Dazao strain, which the main factors are leading to the yellow-green cocoon of Dazao.

Standard method for detecting Bombyx mori nucleopolyhedrovirus disease-resistant silkworm varieties

ABSTRACT Bombyx mori nucleopolyhedrovirus (BmNPV) disease is one of the most serious silkworm diseases, and it has caused great economic losses to the sericulture industry. So far, the disease has not been controlled effectively by therapeutic agents. Breeding resistant silkworm varieties breeding may be an effective way to improve resistance to BmNPV and reduce economic losses. A precise resistance-detection method will help to accelerate the breeding process. For this purpose, here we described the individual inoculation method (IIM). Details of the IIM include pathogen BmNPV preparation, mulberry leaf size, pathogen volume, rearing conditions, course of infection, and breeding conditions. Finally, a resistance comparison experiment was performed using the IIM and the traditional group inoculation method (GIM). The incidence of BmNPV infection and the within-group variance results showed that the IIM was more precise and reliable than the GIM.

Comparison of the Dissolution Performance of Chemical Component and Anticonvulsant Effect between Two Application Forms of Bombyx mori / 中国药房

OBJECTIVE：To study the difference of dissolution performance of chemical component and anticonvulsant effect of Bombyx mori decoction and powder taken with water，and to provide reference for the selection of application forms of B. mori. METHODS：The yield of dry extract was determined for decoction and powder biomimetic gastric juice of B. mori. HPLC method was used to detect the content of ammonium oxalate in decoction and powder biomimetic gastric juice of B. mori. The content of protein in decoction and powder biomimetic gastric juice of B. mori was determined with bicinchoninic acid（BCA）method. Mice was divided into normal group（1％Sodium carboxymethylcellulose solution），model group（1％Sodium carboxymethylcellulose solution），positive group （phenytoin，2 mg/kg） and B. mori decoction and powder suspensions high-dose，medium-dose and low-dose groups（0.75，1.5，3 g/kg by crude drug）according to random number table，with 20 mice in each group. After 60 min of intragastric administration，electric stimulation was conducted，and the rate of convulsion in mice was recorded. RESULTS：The yields of dry extract were 22.08％ and 26.40％ in decoction and powder gastric juice of B. mori （P＜0.05）；the contents of ammonium oxalate were 11.22％ and 16.83％ （P＜0.05），and the contents of protein was 3.39％ and 4.92％ （P＜0.01）. Compared with normal group，convulsion rate of mice was increased significantly in model group （P＜0.01）；compared with model group，convulsion rates of mice were decreased significantly in administration groups （P＜0.05 or P＜0.01），and the convulsion rate of mice in B. mori powder suspensions group was lower than decoction group. CONCLUSIONS：The dissolution performance of the chemical component from gastric juice of B. mori powder is better than that of decoction， and the anticonvulsant effect of B. mori powder is better than decoction.

Expression and characterization of juvenile hormone binding protein Bmtol gene in silkworm, Bombyx mori / 生物工程学报

The head of the silkworm is a nerve center and a sense organ, contains antennaes and sensory hair, feels the outside signal, and responds to the external signal delivered to the brain. Juvenile hormone is mainly synthesized and secreted by corpora allata, and it needs to be played with the aid of the hormone binding protein, because the juvenile hormone binding protein is the carrier of juvenile hormone transport and plays a functional in vivo, they have an extremely important function in insects. The objective of this study is to screened and identify a novel BmTOL proteins that it has a conserved structure of the juvenile hormone binding protein family by SilkDB and NCBI database. Its coding gene number is BGIBMGA003404 (GenBank Accession No. KY681053). We also expressed the recombinant protein using the prokaryotic expression system, and then successfully purified the recombinant protein by Ni-NTA chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bmtol was higher expressed in head. Furthermore, the expression level of Bmtol gene was higher in newly exuviated silkworm, and expression level of Bmtol gene was lower from at 3 days 5th instar to 7 days pupa, began to increase after the moth. Immunohistochemistry showed that BmTOL protein was localized in the cortex, antennaes and brain of the head, It may be related to the information transmission of the head, and provides an important source of information for the growth and development of silkworm.

Interaction of Bombyx mori aminopeptidase N and cadherin-like protein with Bacillus thuringiensis Cry1Ac toxin / 生物工程学报

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.

Ampullae infection of the malpighian tubules of Bombyx mori by Alphabaculovirus / Infecção de células da ampola dos túbulos de malpighi de Bombyx mori pelo Alphabaculovirus

Alphabaculovirus is a genus of the entomopathogenic virus, whose species Bombyx mori nucleopolyhedrovirus (BmNPV) infects the silkworm, Bombyx mori, which is an important insect in the sericulture industry. A geographic isolate of BmNPV was identified in the state of Paraná, Brazil. It was infecting B. mori larvae and various organs and target tissues were identified, however, there was no information about the infection of Malpighian tubules (MT). The MT comprises the excretory system of B. mori and acts in the elimination of toxic substances and in hydroelectrolytic homeostasis. Thus, the present study examined the susceptibility and cytopathology of B. mori MT to BmNPV. To this end, hybrid fifth instar larvae were inoculated with a virus suspension at different days post-inoculation (dpi). MT segments were collected and divided into the ampullae, proximal, medial and distal regions. These were processed for light microscopy and transmission electron analysis. The MT regions revealed differences in susceptibility to BmNPV and the ampullae in its transition area was infected from the sixth dpi; the other regions did not reveal any evidence of infection. The transition area of the ampullae has not been previously described in Lepidoptera and its cytopathology revealed a hypertrophic nucleus with viroplasm, followed by the formation and development of viral polyhedra, which are common characteristics of infections by Alphabaculovirus. Thus, infection of the ampullae of the MT of B. mori by BmNPV, together with other known targets, compromises the metabolic balance of the insect, which results in consequences for silk production and damage to the sericulture sector.

Alphabaculovirus é um gênero de vírus entomopatogênico, cuja espécie Bombyx mori nucleopolyhedrovirus (BmNPV) infecta o bicho-da-seda, Bombyx mori, inseto importante na indústria sericícola. Um isolado geográfico do BmNPV foi identificado no estado do Paraná, Brasil, infectando lagartas de B. mori e vários órgãos e tecidos alvos foram identificados; entretanto, não há informações sobre a infecção do túbulo de Malpighi (TM). O TM compõe o sistema excretor de B. mori, atuando na eliminação de substâncias tóxicas e na homeostase hidroeletrolítica. Assim, o presente estudo, analisou a susceptibilidade e a citopatologia do TM de B. mori ao BmNPV. Para tanto, lagartas híbridas de 5° instar foram inoculadas com uma suspensão viral e em diferentes dias pós-inoculação (dpi), segmentos do TM foram coletados e subdivididos nas regiões da ampola, proximal, média e distal; sendo processados para análises em microscopias de luz e eletrônica de transmissão. As regiões do TM revelaram diferenças na susceptibilidade ao BmNPV e a ampola, na sua área de transição, foi infectada a partir do 6° dpi, já as demais regiões não revelaram quaisquer indícios de infecção. A área de transição da ampola, ainda não havia sido descrita em lepidópteros e sua citopatologia revelou núcleo hipertrófico com viroplasma, seguido da formação e desenvolvimento dos poliedros virais, características comuns das infecções pelo Alphabaculovirus. Assim, a infecção da ampola do TM de B. mori ao BmNPV, somada a de outros alvos conhecidos, compromete o equilíbrio metabólico do inseto, com consequências na produção de seda e prejuízos ao setor sericícola.

Infection of silkworm larvae by the entomopathogenic fungus Metarhizium anisopliae / Infecção de lagartas do bicho-da-seda pelo fungo entomopatogênico Metarhizium anisopliae

ABSTRACT: The isolate E9 of Metarhizium anisopliae was used in commercial hybrids of Bombyx mori larvae to evaluate its biological effect. Symptomatological analyses showed typical signs of fungal infection. Histopathology revealed the presence of large numbers of hemocytes in the hemocoel, and on the sixth dpi the bodies of the insects appeared to be colonised by the fungus. The isolate E9 is pathogenic to larvae B. mori and; therefore, death of the insects was caused by the colonization of fungus in the epidermal and mesodermal tissues.

RESUMO: O isolado E9 de Metarhizium anisopliae foi usado em larvas híbridas de Bombyx mori para avaliar seu efeito biológico. A sintomatologia revelou sinais típicos de infecção por fungos. Na histopatologia foi verificado um aumento no número de hemócitos, sendo que, no 6 dpi, todo o corpo do inseto se apresentou colonizado pelo fungo. O isolado E9 é patogênico para lagartas de B. mori, causando sua morte pela colonização dos tecidos de origem epidérmica e mesodérmica.

Prokaryotic expression, purification and characterization of arginine kinase of Bombyx mori / 生物工程学报

Arginine kinase (AK) is a key enzyme in energy metabolism of invertebrates and plays an important regulatory role in the life activities such as growth and development, nutrition utilization, immune resistance and stress response. Arginine kinase of Bombyx mori (BmAK) is related to the energy balance and anti-NPV process, but there is little research on its molecular structure and enzymatic properties. We cloned the ORF sequence of BmAK gene, and analyzed chromosomal localization, genomic structure, mRNA structure, secondary and tertiary structure. Phylogenetic analysis indicated that AK was highly conserved in evolution. Soluble recombinant BmAK was obtained by prokaryotic expression, and purified by Ni-NTA affinity chromatography. The circular dichroism spectroscopy showed that BmAK contained α-helix structures, and its α-helix structures were relatively stable in the pH range between 5 and 10. Enzyme activity analysis showed that the optimum temperature of BmAK was 30 ℃ and the optimum pH of BmAK was 7.5. The optimal temperature of BmAK was 25 ℃. Between 15 ℃ and 30 ℃, the structure and activity of BmAK was relatively stable. The structure of BmAK was relatively stable at pH 7.0. Our findings reveal the structure and function of BmAK to develop novel green safe and environmentally friendly insecticides.

Cloning, expression, purification and structure analysis of protein tyrosine phosphatase of Bombyx mori / 生物工程学报

Protein tyrosine phosphatase (PTP, EC 3.1.3.48) specifically catalyzes the removal of phosphate groups from phosphorylated tyrosine residues, resulting in protein dephosphorylation, thus regulates life activities such as cell growth, proliferation, differentiation and immunization. Protein tyrosine phosphatase h of Bombyx mori (BmPTP-h) is involved in the replication of nucleopolyhedrovirus (NPV) in Bombyx mori, but the structure and properties of BmPTP-h are little known so far. In this study, the coding sequence of BmPTP-h gene was cloned from the midgut of Bombyx mori, and its genomic structure, mRNA structure, sequence signature, secondary structure and the state in solution were analyzed. Homologous amino acid sequences alignment analysis indicated that BmPTP-h had a high similarity to PTP sequences of numbers of insect NPVs, implying that they may have a common ancestor and similar function. We constructed a prokaryotic expression vector, expressed and obtained the soluble recombinant BmPTP-h in Escherichia coli at 25 ℃, and purified BmPTP-h using Ni-NTA affinity chromatography. Gel filtration analysis showed that BmPTP-h was able to form aggregate and monomer in solution. Circular dichroism spectroscopy analysis showed that the recombinant BmPTP-h contained α-helix structure. Increasing temperature resulted in the unfolding of the α-helix structure of BmPTP-h and the decrease of the α-helix structure content of BmPTP-h. These studies provide a basis to better study the structure and regulation mechanism of BmPTP-h.

Identification and characterization of epidermal growth factor receptor ligands in Bombyx mori / 生物工程学报

Epidermal growth factor receptor (EGFR) is a multi-functional receptor distributed throughout the metazoa. Study on its ligands so far remained mainly on mammals, including how ligands are processed into active forms, their interaction with EGFR, and the signaling pathway they induce. However, in invertebrates, ligands are more divergent among species. Currently, except for Drosophila, less is known about the insect EGFR ligands. Here, we identified two EGFR ligands in Bombyx mori by homology search, domain prediction, analysis of the potential translation initiation sequence and construction of phylogenetic tree, termed as BmEGF-1 and BmEGF-2. BmEGF-1 shows the greatest similarity to Drosophila Spitz and their Rhomboid-recognition motifs are highly identical. BmEGF-2 is a homolog to Drosophila Vein. Then we purified BmEGF-1 extracellular domain expressed in E. coli, and performed pull-down assay with BmEGFR extracellular domain secreted by Sf9 cells. The result confirmed their interaction. Lastly, we found the phosphorylation level of ERK and p38 MAPK was elevated after expression of BmEGF-1 in BmE cells, which suggested that BmEGF-1 is not only able to activate the canonical ERK signaling pathway, but may participate in other cellular processes by inducing p38 MAPK signaling pathway. Our study provides reference to further study of the biological function of BmEGF in silkworm.

Interaction of aminopeptidase (BmAPN5) and parasporal crystal (PC) toxin isolated from Bacillus bombysepticus / 生物工程学报

Aminopeptidase N (APN) belonging to zinc-dependent metalloproteinase, not only catalyzes protein proteolytic process, but also is involved in the pathogenic process as the receptor of pathogenic toxin. In Bombyx mori, APN gene family consists of 16 members, of which BmAPN4 binds trypsin-activated parasporal crystal (PC) toxin isolated from Bacillus bombysepticus (Bb). In order to verify whether or not other APNs interact with PC toxin during the pathogenesis of Bb, we cloned BmAPN5, a member of aminopeptidase family, from the silkworm midgut. The full length of BmAPN5 is 3313 bp, encoding 953 amino acids, containing a zinc peptidase_M1 and ERAP1_C domains. A recombinant GST-BmAPN5 was purified by a prokaryotic expression system. Far-Western blotting, co-immunoprecipitation and ELISA. Binding saturation assays demonstrated that PC after activated by trypsin could be bound by BmAPN5. Additionally, cytotoxic activity of trypsin-activated PC in Sf9 cells transfected with BmAPN5 showed that cells exhibited dramatic cytological changes, including swelling and lysis, revealing BmAPN5 serves as a functional receptor that participates in Bb and PC pathogenicity. These provide some clues for further exploring the pathogenesis relationships of Bb and host.