Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.185
S. Jo, Ji‐Hyun Choi, Ju-Il Kang, Jae-hwan Lim, Y. Seok, J. M. Lee, T. Kusakabe, S. Hong
Sun Jung Jo et al. Screening of Domestic Silkworm Strains for Efficient Heterologous Protein Expression by Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) 186 187 strains screened were highly permissive to BmNPV. Materials and Methods Preparation of recombinant baculovirus In this study, a previously constructed (Kawakami et al., 2008) recombinant BmNPV strain, expressing a luciferase reporter gene, was used (Fig. 1). In order to insert the luciferase gene downstream of the polyhedrin promoter of pFastBac1 (Invitrogen, Grand Island, NY, USA), the luciferase gene was amplified from pFastBac1BmActin3 (Lee et al., 2007) using a forward primer 5’-GAGCTCATGGAAGATGCCAAAAA-3’ containing a SacI restriction site and a reverse primer 5’-CCGCCCTTCTTGGCCTTAATGAGA-3’ and then digested with SacI (Takara Bio Inc., Japan). The pFastBac1 was digested with NotI (Takara Bio Inc., Japan), blunt-ended by T4 DNA polymerase (Takara Bio Inc., Japan), and further digested with SacI. The resultant pFastBac1 vector was ligated to the PCR fragment digested by SacI, using T4 DNA ligase (Takara Bio Inc., Japan). BmDH10Bac cells were transformed with the donor vector pFastBac1polH-Luc (Motohashi et al., 2005) by Tn7 transposase-mediated transposition. The recombinant BmNPV/ BmA3-Luc bacmid was isolated with the alkaline lysis method and used for transfection of BmN4 cells (1.5×10 5 cells) by using the FuGENE HD transfection reagent (Promega, Madison, WI, USA). BmN4 cells were then incubated at 25°C in TNM-FH insect medium (Wilgene, Korea) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) for 4 days, after which the supernatant was collected and stored at 4°C after three iterations to achieve final viral titers of 0.5 × 10 5 plaque-forming units (pfu) per milliliter. Silkworms and inoculation The experimental silkworm larvae comprised the larvae of 52 strains and, as a control strain, the F1 hybrid strain 125 × 126 (a certified strain used by farmers during summer and fall), supplied by the Gyeongsangbuk-do and the Kangwon-do Foundation Original Seed Production Center (Sericulture and Entomology Experiment Station), respectively. Details regarding these 52 silkworm strains are described in the National Academy (Dartar et al., 1993; Jarvis et al., 2008), plant (Sterling, 1989; Kusnadi et al., 1997), and insect systems (Maiorella et al., 1988; Morrow, 2007), are available. In these organisms, insect larvae expression systems (Mathavan et al., 1995; Sumathy et al., 1996) as well as insect cells (Possee, 1997; Kost et al., 1999) are among the most widely used systems for the routine production of recombinant proteins. The most commonly used vector systems for recombinant protein expression in insects are baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV), which display host specificity for infection (Mori et al., 1995; Motohashi et al., 2005). The use of insect larvae and pupae as b
随后，每条第一链cDNA产物取1μL进行半实时和定量RT-PCR。每50μL反应混合物含有0.5 μL 1 st cDNA, 0.25 μL 5 U/μL TaKaRa Ex Taq聚合酶(TaKaRa Bio Inc.， Japan)， 5 μL 10X Ex Taq缓冲液，4 μL dNTP混合物，各10 pmol 5 '和3 '引物。热循环曲线如下:94°C 5分钟，94°C 30秒，57°C 30秒，72°C 30秒的27个循环，然后在72°C下延长5分钟(Takara Bio Inc.，日本)。每个PCR产物在1.5%琼脂糖凝胶中在Tris乙酸EDTA (TAE)缓冲液中电泳。接下来，根据SYBR Premix Ex Taq (Takara Bio Inc.，日本)在Applied Biosystems 7500 real-time PCR系统(Applied Biosystems, Grand Island, NY, USA)中进行实时PCR。热循环曲线如下:在94℃下变性30 s，然后在95℃下变性5 s, 60℃下变性34 s，循环40次。以家蚕18S rRNA基因为内参。半定量实时RT-PCR的正向引物和反向引物分别为5 ' gtacaccttcgtgacttcccattt -3 '和5 ' -AATCCGGTACTGCCACTACTGTTC-3 '，基于荧光素酶序列。18S rRNA(内源对照)的正向引物为5′-GCCTGCGGCTTAATTTGACT-3′，反向引物为5′-CAACTAAGAACGGCCATGCA-3′。将回收的脂肪体(每只家蚕幼虫可收获0.3 g脂肪体)悬浮在1 mL匀浆缓冲液中(0.5 M Tris缓冲液，含0.5 M NaCl, 1% NP40, 5% complete, pH 8.0)，使用Vibra-Cell匀浆器(sonic，美国)在4°C下匀浆5分钟。匀浆在4℃下14000 rpm超离心20 min，上清液重新离心，用于western blotting和ELISA分析。用牛血清白蛋白作为标准品，用比辛胆酸(BCA)法测定纯化蛋白的浓度(Smith et al.， 1985)。蚕蛹脂肪体样品在4倍Laemmli样品缓冲液中变性，在10% SDS-PAGE上分离。电泳后，将蛋白转移到PVDF膜上进行western blotting (Millipore, USA)，并将膜在含5%脱脂牛奶的TBS-0.1% Tween 20 (TBST)中室温(RT)封闭1小时，用Agricultural Sciences ' Genebank Information Center ' (http:// www.genebank.go.kr/)洗涤。本研究使用的9个杂交种是通过本研究第三次筛选筛选出的高容性株系随机杂交而成，并保存在江原道基金会原种生产中心。在25°C ~ 27°C条件下，用新鲜桑叶饲养家蚕，用注射器(Hamilton Co.， Reno, NV, USA)将5龄幼虫注射到血液中，注射20 μL BmNPV/BmA3-Luc杆状病毒(0.5 × 10 5 pfu/mL)。注射后第4天采集家蚕幼虫血淋巴，加入1%苯硫脲(Sigma-Aldrich, St. Louis, MO, USA)防止黑化。收集血淋巴后，将幼虫解剖，保存于- 70°C后进行实验。将收集到的血淋巴在3000 rpm下离心10 min，去除上清，用磷酸盐缓冲盐水(PBS, pH 7.3)洗涤血细胞，然后在120 μL含有25 mM三磷酸(pH 7.8)、2 mM 1,2-二胺环己烷-N,N '，N'-四乙酸(DTT)、2 mM 1,2环己烷二胺四乙酸一水合物(CDTA)、10%甘油和1% Triton X-100的裂解缓冲液中重悬约10 min。共将100 μL的裂解液加入等量的含有甲虫荧光素的底物溶液中，用GloMax Luminometer (Promega, USA)测定荧光素酶活性。数据以每微克总细胞蛋白的相对光单位(RLU)表示。转录分析利用TRIzol (Invitrogen公司，Grand Island, NY, USA)进行荧光素酶测定，从每个家蚕品系的脂肪体中分离总RNA，并用无RNA酶的水洗脱。利用日本第一款cDNA合成试剂盒(Toyobo, Japan)将每个家蚕品系的总RNA 0.5 μg逆转录为cDNA，进行半定量反转录PCR (RTPCR)分析。20 μL反应混合物，含0.5 μg总RNA、4 μL 5X逆转录酶缓冲液、2 μL 10 mM dNTPs、1μL 10 U/ μL RNase抑制剂、1μL 10 pmol/μL oligo (dT)20引物和1μL ReverTra Ace®逆转录酶。家蚕核型多角体病毒(Bombyx mori Nuclear Polyhedrosis Virus, BmNPV) 188189 (OD)高效表达家蚕品系的筛选。以萤火虫荧光素酶参比血清(Sigma, USA, 1 mg/mL)作为荧光素酶标品。利用BmNPV技术选择高容性家蚕品系作为生物反应器。
{"title":"Screening of Domestic Silkworm Strains for Efficient Heterologous Protein Expression by Bombyx mori Nuclear Polyhedrosis Virus (BmNPV)","authors":"S. Jo, Ji‐Hyun Choi, Ju-Il Kang, Jae-hwan Lim, Y. Seok, J. M. Lee, T. Kusakabe, S. Hong","doi":"10.7852/IJIE.2014.29.2.185","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.185","url":null,"abstract":"Sun Jung Jo et al. Screening of Domestic Silkworm Strains for Efficient Heterologous Protein Expression by Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) 186 187 strains screened were highly permissive to BmNPV. Materials and Methods Preparation of recombinant baculovirus In this study, a previously constructed (Kawakami et al., 2008) recombinant BmNPV strain, expressing a luciferase reporter gene, was used (Fig. 1). In order to insert the luciferase gene downstream of the polyhedrin promoter of pFastBac1 (Invitrogen, Grand Island, NY, USA), the luciferase gene was amplified from pFastBac1BmActin3 (Lee et al., 2007) using a forward primer 5’-GAGCTCATGGAAGATGCCAAAAA-3’ containing a SacI restriction site and a reverse primer 5’-CCGCCCTTCTTGGCCTTAATGAGA-3’ and then digested with SacI (Takara Bio Inc., Japan). The pFastBac1 was digested with NotI (Takara Bio Inc., Japan), blunt-ended by T4 DNA polymerase (Takara Bio Inc., Japan), and further digested with SacI. The resultant pFastBac1 vector was ligated to the PCR fragment digested by SacI, using T4 DNA ligase (Takara Bio Inc., Japan). BmDH10Bac cells were transformed with the donor vector pFastBac1polH-Luc (Motohashi et al., 2005) by Tn7 transposase-mediated transposition. The recombinant BmNPV/ BmA3-Luc bacmid was isolated with the alkaline lysis method and used for transfection of BmN4 cells (1.5×10 5 cells) by using the FuGENE HD transfection reagent (Promega, Madison, WI, USA). BmN4 cells were then incubated at 25°C in TNM-FH insect medium (Wilgene, Korea) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) for 4 days, after which the supernatant was collected and stored at 4°C after three iterations to achieve final viral titers of 0.5 × 10 5 plaque-forming units (pfu) per milliliter. Silkworms and inoculation The experimental silkworm larvae comprised the larvae of 52 strains and, as a control strain, the F1 hybrid strain 125 × 126 (a certified strain used by farmers during summer and fall), supplied by the Gyeongsangbuk-do and the Kangwon-do Foundation Original Seed Production Center (Sericulture and Entomology Experiment Station), respectively. Details regarding these 52 silkworm strains are described in the National Academy (Dartar et al., 1993; Jarvis et al., 2008), plant (Sterling, 1989; Kusnadi et al., 1997), and insect systems (Maiorella et al., 1988; Morrow, 2007), are available. In these organisms, insect larvae expression systems (Mathavan et al., 1995; Sumathy et al., 1996) as well as insect cells (Possee, 1997; Kost et al., 1999) are among the most widely used systems for the routine production of recombinant proteins. The most commonly used vector systems for recombinant protein expression in insects are baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV), which display host specificity for infection (Mori et al., 1995; Motohashi et al., 2005). The use of insect larvae and pupae as b","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82747876","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.179
Wan-Taek Ju, Kee-Young Kim, Gyoo-Byung Sung, Yong‐Soon Kim
{"title":"Effects of Physiological Active Substance Extracted from Silkworm Fece","authors":"Wan-Taek Ju, Kee-Young Kim, Gyoo-Byung Sung, Yong‐Soon Kim","doi":"10.7852/IJIE.2014.29.2.179","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.179","url":null,"abstract":"","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86616820","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.198
Sohel Ahmad, V. Wornoayporn, P. Rempoulakis, E. Fontenot, I. Haq, C. Cáceres, H. Paulus, M. Vreysen
The olive fly Bactrocera oleae (Rossi) is the key pest for olive cultivation worldwide. Substantial effort has been invested in the development of the sterile insect technique (SIT) to control this pest. One of the limitations to develop SIT technology for olive fruit fly is the low ability of wild females to lay eggs in other medium than olive fruits, and their slow adaptation to oviposition in artificial substrates. In the present study, fruit grapes were used as an alternative egg collection medium to harvest eggs and young larvae from freshly colonized wild strains originating from France, Italy, Spain and Croatia. The larvae were allowed to develop into the fruits until the second instar, before they were extracted out and further reared on a standard artificial diet. Furthermore, F1 to F4 female flies were alternatively offered wax bottles to oviposit. Finally, the performance of hybrid strains created from crosses between wild and long colonised flies was assessed. The results showed that females of all 4 wild strains readily oviposited eggs in grapes and from the F2 generation onward, females from all strains were adapted to laying eggs in wax bottles. No difference was observed in eggs and pupae production among all strains tested. The findings are discussed for their implications on SIT application against olive fruit fly.
{"title":"Hybridization and Use Of Grapes as an Oviposition Substrate Improves the Adaptation of Olive Fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) to Artificial Rearing Conditions","authors":"Sohel Ahmad, V. Wornoayporn, P. Rempoulakis, E. Fontenot, I. Haq, C. Cáceres, H. Paulus, M. Vreysen","doi":"10.7852/IJIE.2014.29.2.198","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.198","url":null,"abstract":"The olive fly Bactrocera oleae (Rossi) is the key pest for olive cultivation worldwide. Substantial effort has been invested in the development of the sterile insect technique (SIT) to control this pest. One of the limitations to develop SIT technology for olive fruit fly is the low ability of wild females to lay eggs in other medium than olive fruits, and their slow adaptation to oviposition in artificial substrates. In the present study, fruit grapes were used as an alternative egg collection medium to harvest eggs and young larvae from freshly colonized wild strains originating from France, Italy, Spain and Croatia. The larvae were allowed to develop into the fruits until the second instar, before they were extracted out and further reared on a standard artificial diet. Furthermore, F1 to F4 female flies were alternatively offered wax bottles to oviposit. Finally, the performance of hybrid strains created from crosses between wild and long colonised flies was assessed. The results showed that females of all 4 wild strains readily oviposited eggs in grapes and from the F2 generation onward, females from all strains were adapted to laying eggs in wax bottles. No difference was observed in eggs and pupae production among all strains tested. The findings are discussed for their implications on SIT application against olive fruit fly.","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81112401","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.214
M. Ahn, Jae‐Sam Hwang, H. Yoon, E. Yun
We prepared antibodies from insect glycosaminoglycans (GAGs) and assayed the titer. Nine polyclonal antibodies against insect GAGs were raised for development of an ELISA in biological fluids (mice serum). The 3th booster collection of antiserum of BALB/c mice as a primarily antibody was assayed for titer determination by ELISA method. In sandwich ELISA of GAGs derived from Isaria sinclairii or other insects, antiserum from insect GAGs gave satisfactory results for so potent antibody(100: 1 ∼ 1000:1) raising (manufacturing) agent in range of 10 ng/ml.
{"title":"Antibody derived from insect glycosaminoglycan","authors":"M. Ahn, Jae‐Sam Hwang, H. Yoon, E. Yun","doi":"10.7852/IJIE.2014.29.2.214","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.214","url":null,"abstract":"We prepared antibodies from insect glycosaminoglycans (GAGs) and assayed the titer. Nine polyclonal antibodies against insect GAGs were raised for development of an ELISA in biological fluids (mice serum). The 3th booster collection of antiserum of BALB/c mice as a primarily antibody was assayed for titer determination by ELISA method. In sandwich ELISA of GAGs derived from Isaria sinclairii or other insects, antiserum from insect GAGs gave satisfactory results for so potent antibody(100: 1 ∼ 1000:1) raising (manufacturing) agent in range of 10 ng/ml.","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77824222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.207
S. Bae, Seung Hee Lee, W. S. Kwak, Y. Ahn, T. Shin, S. Woo
The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein E rns (E0), E1 and E2. Among them, E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. In this study, to determine the optimal expression conditions of glycoprotein E2 using baculovirus system, we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZA DNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21 cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity of infection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) were tested, High-Five cells showed the highest production. In addition, four different serum-free and serum-supplemented media, respectively, were tested for the expression of glycoprotein E2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided the best conditions for protein production and the BV yield.
{"title":"Optimal Conditions for the Expression of Glycoprotein E2 of Classical Swine Fever Virus using Baculovirus in Insect Cells","authors":"S. Bae, Seung Hee Lee, W. S. Kwak, Y. Ahn, T. Shin, S. Woo","doi":"10.7852/IJIE.2014.29.2.207","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.207","url":null,"abstract":"The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein E rns (E0), E1 and E2. Among them, E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. In this study, to determine the optimal expression conditions of glycoprotein E2 using baculovirus system, we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZA DNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21 cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity of infection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) were tested, High-Five cells showed the highest production. In addition, four different serum-free and serum-supplemented media, respectively, were tested for the expression of glycoprotein E2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided the best conditions for protein production and the BV yield.","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82944460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.169
W. Hassan, B. S. Nath
Wazid Hassan and B. Surendra Nath Genetic diversity and phylogenetic relationships among microsporidians isolates from the Indian tasar silkworm...... 170 171 Materials and Methods Origin of microsporidian isolates. Twenty two microsporidians were collected from individual infected tasar silkmoths of A. mylitta during the survey conducted from 2010 to 2013 in different geographic reserved forests areas (districts of Giridih, Deoghar, Dumka, Dhanbad, Kharshawan, Chaibasha, WestSinghbhum, East-Singhbhum and Ranchi in Jharkhand State, India (Fig. 1 and Table 1). Microsporidian spores were isolated from infected tasar silkmoths by maceration and suspended them in 0.85% NaCl followed by filtration through cheese cloth and centrifugation at 3500 r/min for 10 min. The spore pellet was Percoll purified by gradient centrifugation as described by Undeen and Alger (1971). Each of the purified microsporidian isolates were maintained in vivo in isolation, through per oral inoculation and designated as MIJ-1sG, MIJ-2bG, MIJ-3gG, MIJ-4mG, MIJ-1jD, MIJ-2pD, MIJ-3sD, MIJ-4cD, MIJ-5mD, MIJ-1kDm, MIJ-1gDn, MIJ-1bR, MIJ-2pR, MIJ-3rR, MIJ-1kK, MIJ-1gC, MIJ-1cWS, MIJ-2mWS, MIJ-3gWS, MIJ-4nWS, MIJ-1cES and MIJ-2dES along with type species NIK-1s_mys. The details of microsporidian isolates, places of collection, host, shape and size are presented in Table 1. Measurement of spore length and width. The morphology of purified spores was observed under phase contrast microscope. The length and width of spores were measured according to the method of Undeen and Vavra (1997). Fresh spores were spread in water agar on glass micro-slides and measured using an At present, tasar silk production has increased considerably; but even after there is huge gap in the production and demand. The important reasons for the low production are attributed to traditional rearing on trees in a natural habitat, which exposes the larvae to a number of diseases caused by microbial pathogens apart from the natural calamities. As tasar silkworm rearing is outdoor in nature, it is affected by several diseases viz., microsporidiosis, virosis, bacteriosis and muscardine. The crop loss in tasar culture due to silkworm diseases varies from 40 50% (Sing et al. 2011). The disease caused by the microsporidian, Nosema bombycis is the most devastating in tasar silkworms and inflicts severe cocoon crop loss and passed onto the next generation transovarially. Microsporidia are a diverse group of spore-forming obligate intracellular parasites including more than 1300 formally described species in 160 genera (Wittner and Weiss 1999, Keeling 2009). Microsporidia are unique eukaryotes, which do not possess centrioles, and mitochondrial apparatus, although nuclei are present in distinct number (Vossbrinck and Woese 1986, Vossbrinck et al. 1987). These parasites infect a wide range of invertebrates and vertebrates including insects, fishes, mammals and protists (Wittner and Weiss 1999, Wasson and Peper 2000, Weiss 20
Wazid Hassan和B. Surendra Nath印度沙蚕微孢子虫分离株的遗传多样性和系统发育关系......微孢子虫分离物的材料与方法。2010 - 2013年，在贾坎德邦不同地理保护区(吉里迪、德奥哈尔、杜姆卡、丹巴德、哈尔沙万、柴巴沙、西辛格布姆、东辛格布姆和兰齐)采集到小孢子虫22只。印度(图1和表1)。从感染的tasar蚕蛾中通过浸渍分离出微孢子孢子，将其悬浮在0.85%的NaCl中，然后用奶酪布过滤，以3500 r/min的速度离心10分钟。按照Undeen和Alger(1971)的描述，用Percoll梯度离心纯化孢子球。每个纯化的微孢子虫分离株通过口服接种在体内保持分离，并被命名为MIJ-1sG、MIJ-2bG、MIJ-3gG、MIJ-4mG、MIJ-1jD、MIJ-2pD、MIJ-3sD、MIJ-4cD、MIJ-5mD、MIJ-1kDm、MIJ-1gDn、MIJ-1bR、MIJ-2pR、MIJ-3rR、MIJ-1kK、MIJ-1gC、MIJ-1cWS、MIJ-2mWS、MIJ-3gWS、MIJ-4nWS、MIJ-1cES和MIJ-2dES以及类型种NIK-1s_mys。微孢子虫分离株、采集地点、寄主、形状和大小的详细情况见表1。测量孢子的长度和宽度。在相差显微镜下观察纯化孢子的形态。孢子的长度和宽度根据Undeen和Vavra(1997)的方法测定。将新鲜孢子散布于玻璃微玻片上的琼脂水中，并利用微玻片进行测量。但即使在生产和需求方面也存在巨大差距。造成产量低的重要原因是传统的在自然栖息地的树上饲养，除了自然灾害外，还使幼虫暴露于许多由微生物病原体引起的疾病。由于柞蚕是在室外饲养的，因此易受微孢子虫病、病毒病、细菌病和蝇蛆病等病害的影响。桑蚕病害造成的tasar栽培作物损失从40 - 50%不等(Sing et al. 2011)。这种由微孢子虫(Nosema bombycis)引起的疾病对沙蚕的危害最大，造成严重的蚕茧损失，并经卵巢遗传给下一代。微孢子虫是一种多样的形成孢子的专性细胞内寄生虫，包括160属1300多种正式描述的物种(Wittner and Weiss 1999, Keeling 2009)。微孢子虫是一种独特的真核生物，它不具有中心粒和线粒体装置，尽管细胞核数量不同(Vossbrinck and Woese 1986, Vossbrinck et al. 1987)。这些寄生虫感染广泛的无脊椎动物和脊椎动物，包括昆虫、鱼类、哺乳动物和原生生物(Wittner and Weiss 1999; Wasson and pepper 2000; Weiss 2001)。根据微孢子虫的形态、超微结构、生命周期和寄主-寄生虫关系，发现了大量的微孢子虫新种。基于DNA标记谱的分子系统发育分析在很大程度上克服了与超微结构和基于表型的分类系统相关的问题(Baker et al. 1995, Hartskeerl et al. 1995, Mathis et al. 1997, Hung et al. 1998)。随机扩增多态性DNA-PCR检测使用一组任意核苷酸序列的引物(Welsh and McClelland 1990, Williams et al. 1990)被描述为分析多种生物遗传多样性和系统发育的潜在分子标记系统(Hadrys et al. 1992, Lu and Rank 1996)。RAPD技术已被用于生成分子标记，以确定小虫种/株之间的遗传多样性和系统发育关系(Tsai et al. 2003, Rao et al. 2007, Nath et al. 2011)。本文利用RAPD-PCR技术对印度贾坎德邦不同森林地区分离的塔沙蚕(A. mylitta)微孢子虫的遗传多样性进行了分析。图1所示。贾坎德邦的地图，显示了热带tasar, A. mylitta在九个地理森林区域的分布。Int。j .的尘埃。Entomol。第29卷第1期(2)， pp. 169-178(2014) 170 171。我把这些东西都拿出来，把它们拿出来，把它们拿出来，把它们拿出来，把它们拿出来，把它们拿出来，把它们拿出来，把它们拿出来，把它们拿出来
{"title":"Genetic Diversity and Phylogenetic Relationships among Microsporidian Isolates from the Indian Tasar Silkworm, Antheraea mylitta, as Revealed by RAPD Fingerprinting Technique","authors":"W. Hassan, B. S. Nath","doi":"10.7852/IJIE.2014.29.2.169","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.169","url":null,"abstract":"Wazid Hassan and B. Surendra Nath Genetic diversity and phylogenetic relationships among microsporidians isolates from the Indian tasar silkworm...... 170 171 Materials and Methods Origin of microsporidian isolates. Twenty two microsporidians were collected from individual infected tasar silkmoths of A. mylitta during the survey conducted from 2010 to 2013 in different geographic reserved forests areas (districts of Giridih, Deoghar, Dumka, Dhanbad, Kharshawan, Chaibasha, WestSinghbhum, East-Singhbhum and Ranchi in Jharkhand State, India (Fig. 1 and Table 1). Microsporidian spores were isolated from infected tasar silkmoths by maceration and suspended them in 0.85% NaCl followed by filtration through cheese cloth and centrifugation at 3500 r/min for 10 min. The spore pellet was Percoll purified by gradient centrifugation as described by Undeen and Alger (1971). Each of the purified microsporidian isolates were maintained in vivo in isolation, through per oral inoculation and designated as MIJ-1sG, MIJ-2bG, MIJ-3gG, MIJ-4mG, MIJ-1jD, MIJ-2pD, MIJ-3sD, MIJ-4cD, MIJ-5mD, MIJ-1kDm, MIJ-1gDn, MIJ-1bR, MIJ-2pR, MIJ-3rR, MIJ-1kK, MIJ-1gC, MIJ-1cWS, MIJ-2mWS, MIJ-3gWS, MIJ-4nWS, MIJ-1cES and MIJ-2dES along with type species NIK-1s_mys. The details of microsporidian isolates, places of collection, host, shape and size are presented in Table 1. Measurement of spore length and width. The morphology of purified spores was observed under phase contrast microscope. The length and width of spores were measured according to the method of Undeen and Vavra (1997). Fresh spores were spread in water agar on glass micro-slides and measured using an At present, tasar silk production has increased considerably; but even after there is huge gap in the production and demand. The important reasons for the low production are attributed to traditional rearing on trees in a natural habitat, which exposes the larvae to a number of diseases caused by microbial pathogens apart from the natural calamities. As tasar silkworm rearing is outdoor in nature, it is affected by several diseases viz., microsporidiosis, virosis, bacteriosis and muscardine. The crop loss in tasar culture due to silkworm diseases varies from 40 50% (Sing et al. 2011). The disease caused by the microsporidian, Nosema bombycis is the most devastating in tasar silkworms and inflicts severe cocoon crop loss and passed onto the next generation transovarially. Microsporidia are a diverse group of spore-forming obligate intracellular parasites including more than 1300 formally described species in 160 genera (Wittner and Weiss 1999, Keeling 2009). Microsporidia are unique eukaryotes, which do not possess centrioles, and mitochondrial apparatus, although nuclei are present in distinct number (Vossbrinck and Woese 1986, Vossbrinck et al. 1987). These parasites infect a wide range of invertebrates and vertebrates including insects, fishes, mammals and protists (Wittner and Weiss 1999, Wasson and Peper 2000, Weiss 20","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79155130","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.193
S. Bae, Young-Soon Jun, T. Shin, S. Woo
Sung Min Bae et al. Likability of Insects 194 195 classified into several groups according to participants’ gender, age, residence, and previous experience in insect-related events.
裴成民等。根据参与者的性别、年龄、居住地和以前与昆虫有关的事件的经历，将他们分为几组。
{"title":"An Analysis of Likability of Insects in Korea","authors":"S. Bae, Young-Soon Jun, T. Shin, S. Woo","doi":"10.7852/IJIE.2014.29.2.193","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.193","url":null,"abstract":"Sung Min Bae et al. Likability of Insects 194 195 classified into several groups according to participants’ gender, age, residence, and previous experience in insect-related events.","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78515066","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.162
Wan-Taek Ju, Kee-Young Kim, Gyoo-Byung Sung, Yong‐Soon Kim
1-Deoxynojirimycin(1-DNJ or DNJ), a component in silkworm powder, prevents glucose from being absorbed into the bloodstream from the small intestine by inhibiting -glucosidase activity. This study compared the functional components of 1-DNJ from various silkworm species using a gene database with those of 1-DNJ produced by silkworms bred through cross-combinations. We utilized comparisons of geographical origins and species of silkworms using a gene database and discovered that 1-DNJ activity was ranked in the following order by species, Japanese (SK-1) > Chinese (C48) > European (Rock191). 1-DNJ constituted varying percentages of silkworm organs in the following order, blood > epithelial tissue > body fat > silk glands. With regard to sex, 1-DNJ, levels were higher in males than females. However, 1-DNJ levels with respect to various genetic traits (blood color, silk color, and egg color) were consistent. In order to study 1-DNJ changes that occurred during cross breeding of the silkworm gene, we bred cross-combinations utilizing SK-1 and C48 silkworms. In conclusion, in order to provide information about the constituents of functional materials contained in silkworm powder, it is imperative that silkworm cross breeding occurs so that the database of functional materials extracted from silkworms will expand.
{"title":"Quantitative Analysis of 1-Deoxynojirimycin Content Using Silkworm Genetic Resources","authors":"Wan-Taek Ju, Kee-Young Kim, Gyoo-Byung Sung, Yong‐Soon Kim","doi":"10.7852/IJIE.2014.29.2.162","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.162","url":null,"abstract":"1-Deoxynojirimycin(1-DNJ or DNJ), a component in silkworm powder, prevents glucose from being absorbed into the bloodstream from the small intestine by inhibiting -glucosidase activity. This study compared the functional components of 1-DNJ from various silkworm species using a gene database with those of 1-DNJ produced by silkworms bred through cross-combinations. We utilized comparisons of geographical origins and species of silkworms using a gene database and discovered that 1-DNJ activity was ranked in the following order by species, Japanese (SK-1) > Chinese (C48) > European (Rock191). 1-DNJ constituted varying percentages of silkworm organs in the following order, blood > epithelial tissue > body fat > silk glands. With regard to sex, 1-DNJ, levels were higher in males than females. However, 1-DNJ levels with respect to various genetic traits (blood color, silk color, and egg color) were consistent. In order to study 1-DNJ changes that occurred during cross breeding of the silkworm gene, we bred cross-combinations utilizing SK-1 and C48 silkworms. In conclusion, in order to provide information about the constituents of functional materials contained in silkworm powder, it is imperative that silkworm cross breeding occurs so that the database of functional materials extracted from silkworms will expand.","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90142733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.153
Seong‐Jin Hong, Sunyoung Kim, Nergui Ravzanaadii, Kyoungha Han, Seonghyun Kim, N. Kim
Seong-Jin Hong et al. Characteristics of the swallowtail butterfly 154 155 sequestration in the body (Omura et al., 2006). For instance, larvae secrete odoriferous fluid from their defensive glands (osmeteria), which contain five of the eight aristolochic acid compounds found in the host, A. debilis (Nishida, 1995). S. montela is dimorphic at the adult stage, which is characteristic of primitive insects. In the wild, it is simple to distinguish males and females by the color of their wings. Male wings are bright beige, whereas females have dark brown wings that are described as a melanized pattern (Nam et al., 1998), which has not yet been proven. Like most other butterflies, S. montela adults are solitary in behavior. Females lay clutches containing dozens to a hundred eggs on the stems or leaves of the host plant (Shin, 1974). After 5–8 d, larvae hatch from the eggs in a group and pass through five instars with a total developmental time of 16–21 d, depending on the ambient thermal environment and availability of host plants in Korea. Adults survive for about two weeks after pupae metamorphogenesis, which takes 10–12 d unless they enter diapause for overwintering. Taken together, adult emergence takes approximately one month, and completion of the full life cycle lasts up to about 50 d in the field. However, Russian subspecies take 16–19 d for the adult to develop at 20–25°C in the laboratory (Monastyrskiy and Kotlobay, 1995). Therefore, S. montela is multivoltine, emerging two or three times a year on average in Korea (Shin, 1974; Kim and Lee, 1992; Nam et al., 1998). The habitat of S. montela in the Gwangneung area, one of the most important sites for insect conservation in Korea, was investigated for a long period of time from 1932 to 2004 (Shin, 1974; Byun et al., 2005). Swallowtail emergence was observed three times a year from 1960 to 1962 (Shin, 1974). Since then, the population has decreased rapidly until only a single male was observed in April 2004, despite the presence of 148 species (67.0% of the S. Korea butterfly fauna) listed in Gwangneung Forest (Byun et al., 2005). As a part of the ecosystem, the world’s human population has continuously increased, and it predominantly occupies most of the earth while simultaneously destroying or fragmenting natural habitats, which threatens worldwide biodiversity (Fahrig, 2003; Kim, 2010). Global warming caused by the greenhouse effect is a pernicious threat to swallowtails (Collins and Morris, 1985; Collins, 1991), and many species, including S. montela, are facing environmental challenges or further threats. Particularly, because S. montela habitats are intimately associated with human-occupied areas, the species is exposed to additional variety of butterfly specimens, especially swallowtails, have been on display all over the world, and live butterflies are exhibited in indoor gardens and museums for educational and aesthetic purposes. A representative flight mode of butterflies is flutteri
{"title":"Temperature-Dependent Development of the Swallowtail Butterfly, Sericinus montela Gray","authors":"Seong‐Jin Hong, Sunyoung Kim, Nergui Ravzanaadii, Kyoungha Han, Seonghyun Kim, N. Kim","doi":"10.7852/IJIE.2014.29.2.153","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.153","url":null,"abstract":"Seong-Jin Hong et al. Characteristics of the swallowtail butterfly 154 155 sequestration in the body (Omura et al., 2006). For instance, larvae secrete odoriferous fluid from their defensive glands (osmeteria), which contain five of the eight aristolochic acid compounds found in the host, A. debilis (Nishida, 1995). S. montela is dimorphic at the adult stage, which is characteristic of primitive insects. In the wild, it is simple to distinguish males and females by the color of their wings. Male wings are bright beige, whereas females have dark brown wings that are described as a melanized pattern (Nam et al., 1998), which has not yet been proven. Like most other butterflies, S. montela adults are solitary in behavior. Females lay clutches containing dozens to a hundred eggs on the stems or leaves of the host plant (Shin, 1974). After 5–8 d, larvae hatch from the eggs in a group and pass through five instars with a total developmental time of 16–21 d, depending on the ambient thermal environment and availability of host plants in Korea. Adults survive for about two weeks after pupae metamorphogenesis, which takes 10–12 d unless they enter diapause for overwintering. Taken together, adult emergence takes approximately one month, and completion of the full life cycle lasts up to about 50 d in the field. However, Russian subspecies take 16–19 d for the adult to develop at 20–25°C in the laboratory (Monastyrskiy and Kotlobay, 1995). Therefore, S. montela is multivoltine, emerging two or three times a year on average in Korea (Shin, 1974; Kim and Lee, 1992; Nam et al., 1998). The habitat of S. montela in the Gwangneung area, one of the most important sites for insect conservation in Korea, was investigated for a long period of time from 1932 to 2004 (Shin, 1974; Byun et al., 2005). Swallowtail emergence was observed three times a year from 1960 to 1962 (Shin, 1974). Since then, the population has decreased rapidly until only a single male was observed in April 2004, despite the presence of 148 species (67.0% of the S. Korea butterfly fauna) listed in Gwangneung Forest (Byun et al., 2005). As a part of the ecosystem, the world’s human population has continuously increased, and it predominantly occupies most of the earth while simultaneously destroying or fragmenting natural habitats, which threatens worldwide biodiversity (Fahrig, 2003; Kim, 2010). Global warming caused by the greenhouse effect is a pernicious threat to swallowtails (Collins and Morris, 1985; Collins, 1991), and many species, including S. montela, are facing environmental challenges or further threats. Particularly, because S. montela habitats are intimately associated with human-occupied areas, the species is exposed to additional variety of butterfly specimens, especially swallowtails, have been on display all over the world, and live butterflies are exhibited in indoor gardens and museums for educational and aesthetic purposes. A representative flight mode of butterflies is flutteri","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73483606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-31DOI: 10.7852/IJIE.2014.29.2.145
G. Lekha, Esvaran Vijayagowri, Sasibhushan Sirigineedi, V. Sivaprasad, K. Ponnuvel
The variation in the level of immune response related gene expression in silkworm, Bombyx mori following infection with Bombyx mori nucleopolyhedrovirus (BmNPV) was analyzed at different time intervals. The occlusion bodies of BmNPV orally inoculated to the two most divergent silkworm races viz., Sarupat (resistant to BmNPV infection) and CSR2 (susceptible to BmNPV infection) were subjected to oral BmNPV inoculation. The expression profile of gp41 gene of BmNPV in the Sarupat and CSR2 races revealed that the virus could invade the midguts of both susceptible and resistant races. However, its multiplication was significantly less in the midgut of resistant race, while, in the susceptible race, the viral multiplication reached maximum level within 12 h. These findings indicate that potential host genes are involved in the inhibition of viral multiplication within larval midgut. The immune response genes arylphorin, cathepsin B, gloverin, lebocin, serpin, Hsp 19.9, Hsp 20.1, Hsp 20.4, Hsp 20.8, Hsp 21.4, Hsp 23.7, Hsp 40, Hsp 70, Hsp90 revealed differential level of expression on NPV infection. The gloverin, serpin, Hsp 23.7 and Hsp 40 genes are significantly up-regulated in the resistant race after NPV infection. The early up-regulation of these genes suggests that these genes could play an important role in baculovirus resistance in the silkworm, B. mori. 29(2), 145-152
{"title":"Differential Level of Host Gene Expression Associated with Nucleopolyhedrovirus Infection in Silkworm Races of Bombyx mori","authors":"G. Lekha, Esvaran Vijayagowri, Sasibhushan Sirigineedi, V. Sivaprasad, K. Ponnuvel","doi":"10.7852/IJIE.2014.29.2.145","DOIUrl":"https://doi.org/10.7852/IJIE.2014.29.2.145","url":null,"abstract":"The variation in the level of immune response related gene expression in silkworm, Bombyx mori following infection with Bombyx mori nucleopolyhedrovirus (BmNPV) was analyzed at different time intervals. The occlusion bodies of BmNPV orally inoculated to the two most divergent silkworm races viz., Sarupat (resistant to BmNPV infection) and CSR2 (susceptible to BmNPV infection) were subjected to oral BmNPV inoculation. The expression profile of gp41 gene of BmNPV in the Sarupat and CSR2 races revealed that the virus could invade the midguts of both susceptible and resistant races. However, its multiplication was significantly less in the midgut of resistant race, while, in the susceptible race, the viral multiplication reached maximum level within 12 h. These findings indicate that potential host genes are involved in the inhibition of viral multiplication within larval midgut. The immune response genes arylphorin, cathepsin B, gloverin, lebocin, serpin, Hsp 19.9, Hsp 20.1, Hsp 20.4, Hsp 20.8, Hsp 21.4, Hsp 23.7, Hsp 40, Hsp 70, Hsp90 revealed differential level of expression on NPV infection. The gloverin, serpin, Hsp 23.7 and Hsp 40 genes are significantly up-regulated in the resistant race after NPV infection. The early up-regulation of these genes suggests that these genes could play an important role in baculovirus resistance in the silkworm, B. mori. 29(2), 145-152","PeriodicalId":14140,"journal":{"name":"International journal of industrial entomology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80175289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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