A simplified method for the characterization of nuclear polyhedrosis virus genomes

A simplified restriction endonuclease analysis procedure is described which allows the characterization of baculovirus DNA obtained directly from a single larvae without purification of virus. This rapid method was used to demonstrate the genomic stability of nuclear polyhedrosis viruses (NPVs) from Agrotis segetum, Euxoa messoria and Mamestra brassicae after several passages in Euxoa scandens.     

Établissement d'une nouvelle lignée cellulaire (IAFEs-1) a partir d'ovarioles d'Euxoa scandens [Lep.: Noctuidae]

Résumé Une nouvelle lignée cellulaire stable (IAFEs-1) des ovarioles de chrysalides du lepidoptèreEuxoa scandens Riley a été obtenue. La forme prédominante des cellules est sphérique et le doublement de la population cellulaire s'effectue en 18 et 24 h en utilisant respectivement 20 et 10% de sérum de veau foetal. Cette lignée est sensible à divers virus des polyédroses nucléaires et cytoplasmiques.

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Summary A new continuous cell line designated IAFEs-1 was obtained from ovaries ofEuxoa scandens Riley in order to study the replication and the productionin vitro of viruses infecting this cutworm. Predominant cell morphology is spherical and cell population doubling time is 18 and 24 h using respectively 20 and 10% fetal calf serum. Susceptiblity of the cell line to several nuclear and cytoplasmic polyhedrosis viruses is demonstrated.

     

Characterization of entomopox virions of the army cutworm,Euxoa auxiliaris (Lepidoptera: Noctuidae)

Virions were released from virus-containing inclusions (VCI) of an entomopoxvirus of the army cutworm, Euxoa auxiliaris, with carbonate-thioglycolate solution. Knoblike projections present on the surface of the viral envelope were removed by digestion with trypsin. Trypsin-treated virions were homogeneous in both sucrose and CsCl gradients. The virions were similar to vertebrate poxviruses in morphology, contained 1.13 ± 0.3% DNA and had a buoyant density of 1.261 ± 0.003 gm/cm³ in CsCl. The virion preparations were infective and possessed RNA polymerase activity. Of eight species of Lepidoptera tested, only the species from which the virus was originally isolated proved susceptible to infection.     

The effect of hybridization on the periodicity of sex pheromone release of Euxoa species (Lepidoptera: Noctuidae)

ABSTRACT. The periodicities of sex pheromone release by reciprocal F₁ hybrid females obtained from laboratory matings between Euxoa declarata (Walker) and E. rockburnei Hardwick were found to be dependent upon the parental cross. The period of sex pheromone release by females from the E. declarata (female) × E. rockburnei (male) cross paralleled that of the maternal parent species, E. declarata. However, female hybrid E. rockburnei (female) × E. declarata (male) had a prolonged calling period which intersected the pheromone release periods of both parental species and peaked later than that of E. declarata. These results indicate that the circadian periodicity of calling in these species is controlled by autosomal genes and a matroklinal inheritance factor.     

An enzyme-linked immunosorbent assay for the detection of cytoplasmic polyhedrosis virus

An enzyme-linked immunosorbent assay (ELISA) for the detection of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV) is described. Antisera to EsCPV, produced in rabbits and guinea pigs, are specific to EsCPVs when used in an indirect assay. This indirect assay approach permits the detection of homologous antigens at a concentration of about 1 μg/ml; however, this procedure is not suitable to test large numbers of unpurified specimens. For this type of analysis we used a double antibody sandwich assay which can detect 10 ng/ml of homologous antigen in unpurified material without nonspecific reactions. This assay is used to diagnose EsCPV infections in field and laboratory studies.     

Molecular weight of DNA from four entomopoxviruses determined by electron microscopy.

DNA was isolated from entomopoxviruses infected Amsacta moorei and Euxoa auxiliaris (Lepidoptera), Goeldichironomus holoprasinus (Diptera), and Othnonius batesi (Coleoptera) and compared with vertebrate virus DNA (vaccinia). After incubation in Pronase, sodium lauryl sulfate, and deoxycholate, poxvirus preparations shadowed with platinum and palladium revealed subcore particles 45 to 60 nm in diameter. Continued incubation in Pronase resulted in the gradual release of DNA from the particles. Metal-shadowed DNA molecules were photographed in the electron microscope and measured, and the average molecular weights were calculated. Lepidopteran poxvirus DNA (135 X 10(6)) was approximately equal to vaccinia DNA (131.7 X 10(6)) in molecular weight. The molecular weight of dipteran and coleopteran poxvirus DNA (200 X 10(6) to 251 X 10(6)) was approximately 50% greater than vaccinia DNA. Based on the concentration of DNA and protein per virion, Amsacta entomopoxvirus contained 5.7 to 7.7% DNA.     

Detection of DNA base sequence homology between entomopoxviruses isolated from lepidoptera and orthoptera

The extent of DNA base sequence homology between entomopoxviruses (EPVs) from Lepidoptera, Orthoptera, and the vertebrate poxvirus Vaccinia was investigated by DNA-DNA hybridization. α-³²P-Labeled DNA from Amsacta moorei EPV, Melanoplus sanguinipes EPV, and Vaccinia virus strain WR was hybridized with the DNA from six different entomopoxvirus isolates. Based on the thermal denaturation temperature of hybrid DNA molecules, approximately 54% base sequence homology was detected between Amsacta moorei and Euxoa auxiliaris EPV DNAs. Extensive DNA hybridization was detected between α-³²P-labeled Melanoplus sanguinipes EPV DNA and DNA from Arphia conspirsa and Phoetaliotes nebrascensis entomopoxviruses. No base sequence homology was detected between vaccinia DNA and DNA from any of the entomopoxvirus isolates used in this study.     

A nonoccluded virus of the army cutworm

A nonoccluded virus was isolated from larvae of the army cutworm, Euxoa auxiliaris. Infected larvae became lethargic and shrunken, and death usually occurred 12–20 days after infection. The primary site of viral infection and replication appeared to be the nuclei of midgut epithelial cells; however, virus replication also occurred in cells of the tracheal matrix and in muscle. Nuclei in early stages of the infection contained large granular areas with the chromatin scattered near the nuclear membrane. These areas differentiated into viral particles that measured 24 nm and formed crystalline arrays, occasionally 10 μm long. Disruption of the nuclear membrane liberated these arrays of particles into the cytoplasm. Fluorescence microscopy studies indicated that the viral particles contained DNA. The crystalline arrays were Feulgen positive. The virus also infected larvae of the armyworm, Pseudaletia unipuncta, and corn carworm, Heliothis zea, in laboratory tests.     

Improved resolution of the meiotic chromsomes in both sexes of Euxoa species and their hybrids (Lepidoptera: noctuidae).

An air drying and Giemsa staining technique produces consistently good quality cytological preparations when applied to different species of Euxoa (Lepidoptera: Noctuidae) and, in particular, it allows an imporved resolution of the meiotic chromosomes in both sexes. All species so far investigated have a basic haploid chromosome complement of n (male) = 31(XX): n(female) = (XY). A single chiasma per individual bivalent is clearly visible in the male sex. Some chiasmata are formed in interstitial positions, but, by metaphase I, they have all undergone complete terminalization while the bivalents orient axially on the first division spindle. Direct evidence for lack of chiasma formation in the 31 hormorphic bivalents in the female sex is reported for the first time for species of Noctuidae. Evidence in favor of an XY system in the female sex is discussed. Preliminary studies of the meiotic divisions in hybrid material reveal the presence of cryptic structural differences between certain taxa, and will help to elucidate taxonomic problems within this complex genus.     

Structural proteins of Amsacta moorei,Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses

The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine.     

Protein Synthesis in a Lymantria dispar Cell Line Infected by Cytoplasmic Polyhedrosis Virus

The efficiency of replication of a cytoplasmic polyhedrosis virus isolated from a member of the order Lepidoptera, Euxoa scandens, was studied in eight different lepidopterean cell lines. Lymantria dispar cells, which were found to support viral replication, more efficiently, were used to follow the kinetics of appearance of viral-specific polypeptides by a 2-h pulse with [³⁵S]methionine. Five polypeptides (ca. 120,000 molecular weight [120K], 105K, 66K, 46K, and 28K) were identified as components of the polyhedral inclusion bodies, and two polypeptides (112K and 39K) were assigned as viral-particle polypeptides. All these polypeptides were present after 24 h and were still being produced 96 h after infection. The rate of synthesis of the major polyhedral polypeptide (28K) increased in the time course of infection, whereas the background of cellular polypeptides seemed to be unaffected. An indirect immunoperoxidase technique, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was blotted to a nitrocellulose membrane, showed that traces of the major polyhedral polypeptide were found from 8 h postinfection.     

Polyèdres du CPV d’Euxoa scandens [Lep.: Noctuidae] produitsin vivo et sur cellules cultivéesin vitro Études comparatives

Résumé Les polyèdres produits dans les cellules deLymantria dispar L. cultivéesin vitro, après infection par le virus de la polyédrose cytoplasmique d’Euxoa scandens Riley sont de forme exclusivement cubique. Par contre, les formes parasphériques sont rencontrées en très grande majorité lors de l’infection des larves d’E. scandens. La forme cubique se maintient en culture cellulaire après plusieurs passages du virus alors que les polyèdres extraits des cellules infectéesin vitro induisent à nouveauin vivo des inclusions virales de forme hétérogène. Les polyèdres provenent d’un 1^er passage du virusin vitro dans des cellules différentes de celles de l’h?te d’origine entra?nent chez ce dernier une infection causant avec une intensité égle des effets caractéristiques d’une polyédrose cytoplasmique. Néanmoins, les taux d’infection larvaire sont inférieurs à ceux causés par les polyèdres produitsin vivo.

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Summary Cytoplasmic polyhedra first isolated from the larvae ofEuxoa scandens are heterogenous in shape (more than 99% spheric, less than 1% cubic). The non occluded or occluded viruses extracted from infected midgut induce polyhedra, exclusively cubical in shape, inLymantria dispar cells cultivatedin vitro. This shape is maintained after passages of the virus in these cells. Reinfection of the larvae by the polyhedra producedin vitro is characterized by the reapparition of the heterogenous in shape polyhedra. Inclusion bodies synthetized after a 1 st passage of CPVin vitro are less infectious to the original host according to the number of infected larvae. But on the other hand, the physiological effects on the insect of an infection by a cytoplasmic polyhedrosis virus are similar by using either thein vivo orin vitro produced polyhedra.

     

Characterization of the DNA and structural proteins of entomopoxviruses from Melanoplus sanguinipes,Arphia conspirsa, and Phoetaliotes nebrascensis (Orthoptera)

Entomopoxvirus (EPV) occlusion bodies were isolated from virus infected nymphs of the grasshoppers Melanoplus sanguinipes, Arphia conspirsa, and Phoetaliotes nebrascensis. Separation of the viral structural proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave unique protein patterns for each of the three viruses. An occlusion body protein of approximately 100,000 MW was isolated from each virus. Cleavage of viral DNA with HinddIII and BamHI restriction endonucleases and separation of the fragments by agarose gel electrophoresis gave different DNA fragment patterns for each of the three entomopoxviruses. Molecular weight estimates of 120 × 10⁶ for M. sanguinipes EPV DNA, 129 × 10⁶ for A. conspirsa EPV DNA, and 125 × 10⁶ for P. nebrascensis EPV DNA were calculated from the sizes of the viral DNA fragments. Approximately 55% base sequence homology was detected by Southern hybridization of α-³²P-labeledM. sanguinipes EPV DNA with P. nebrascensis DNA. No base sequence homology was detected by Southern hybridization of labeled M. sanguinipes EPV DNA to Othnonius batesi EPV DNA (Coleoptera), Amsacta moorei EPV DNA (Lepidoptera), Euxoa auxiliaris EPV DNA (Lepidoptera), and vaccinia virus DNA fragments.     

Lepidoptera and associated parasitoids attacking Hass and non-Hass avocados in Guatemala

A 5-mo survey for fruit feeding Lepidoptera attacking Hass and non-Hass avocados (Persea americana Miller [Lauraceae]) was conducted in Guatemala from 1 November 2006 to 1 April 2007. In total, 6,740 fruit were collected from 22 different areas in Guatemala. Eight species of Lepidoptera, of which at least two are species new to science, were reared from avocado fruit. Reared Lepidoptera were Amorbia santamaria Phillips and Powell, Cryptaspasma sp. nr. lugubris, Euxoa sorella Schaus, Histura n. sp., Holcocera n. sp., Micrathetis triplex Walker, Netechma pyrrhodelta (Meyrick), and Stenoma catenifer Walsingham. Hymenopteran parasitoids were reared from larvae of C. sp. nr. lugubris and S. catenifer. One species of parasitoid, Pseudophanerotoma sp., was reared from field collected C. sp. nr. lugubris larvae. The dominant parasitoid reared from S. catenifer was a gregarious Apanteles sp. Other parasitoid species reared from S. catenifer larvae were Brachycyrtus sp., Macrocentrus sp., and Pristomerus sp. The oviposition preference of C. sp. nr. lugubris for avocado fruit hanging in trees, dropped fruit on the ground, or exposed avocado seeds was investigated by studying the oviposition preferences of adult female moths and determining egg hatch times in the laboratory, and by investigating the longevity of avocado fruit on the ground under prevailing field conditions. Together, data from these studies suggested that C. sp. nr. lugubris may be an unrecognized pest of avocados that causes hanging fruit to drop to the ground prematurely. The influence of season and altitude on the phenology and distribution of avocado feeding Lepidoptera in Guatemala is discussed.     

Delimitation difficulties in species splits: a morphometric case study on the Euxoa tritici complex (Lepidoptera, Noctuidae)

Abstract. Genital characteristics tend to vary greatly between Lepidoptera species, providing helpful features for species delimitation. The differences between species are usually remarkable and suspicions about species identity never arise. However, fairly often, and possibly increasingly, taxa are elevated to species rank on the basis of very slight morphological differences, often without quantitative support. Euxoa tritici (Linnaeus) is a typical example of a variable species split into several morphologically similar species. The present study tested whether the diagnostic genital characters of the current classification, based on nonquantitative methodology, provide safe identification of species. Both traditional distance morphometrics as well as modern geometric morphometrics, which also enables quantitative shape exploration, were used. Moreover, whether the study specimens can be unambiguously categorized into several species with visual comparisons was tested independently using four specialist entomologists. Genital types of several named species as well as considerable variation in genitalia were found, but no support was found for the presence of several morphologically distinguishable species with quantitative morphometric analyses. Neither were study specimens categorized unambiguously by specialists. The results suggest that pure visual comparisons may lead to unsound taxonomic conclusions and that a quantitative approach in critical cases should be used more frequently.     

农田步甲的生物学研究

本文报道12种农田常见步甲的种群动态和捕食习性,施用杀虫剂对步甲的影响。     

长白山高山苔原带蛾类物种组成及其时间动态

长白山高山苔原带环境条件恶劣,通过对高山苔原带蛾类研究,揭示蛾类物种组成以及时间变化,可为研究蛾类对苔原极端生境的适应能力,以及蛾类在维持苔原带生态平衡中的作用提供依据。2005-2007年和2019年,每年的6、7、8月,在长白山高山苔原带利用灯诱采集蛾类标本,分析蛾类的物种组成以及时间动态。共采集蛾类1585头,隶属于13科126种,夜蛾科（Noctuidae）为优势类群,绿组夜蛾（Anaplectoides prasina）和一色兜夜蛾（Cosmia unicolor）为优势种,稀有种较多。蛾类的种-多度分布接近生态位优先假说。7月份蛾类的物种数、个体数最多,丰富度指数、多样性指数都最高,但均匀度指数却最低。不同种类对时间的反应表现出一定的差异,黄绿组夜蛾（Anaplectoides virens）对8月,厉切夜蛾（Euxoa lidia）对6月的适应力相对较强。各物种的顺序日期存在一定的差异性,只有10种蛾类在3个月份都被采集到。研究表明,长白山高山苔原带蛾类的多样性较低,成虫活跃期较短;不同类群的蛾类在苔原环境中显示出差异化的适应性,夜蛾科的适应能力超过其它类群,尺蛾科（Geometridae）的适应性相对较低,蛾类对时间的变化反应比较敏感。     

Development and characterization of a continuous cell line,AFKM-On-H,from hemocytes of the European corn borer <Emphasis Type="BoldItalic">Ostrinia nubilalis</Emphasis> (Hübner) (Lepidoptera,Pyralidae)

The corn borer, Ostrinia nubilalis, is a very important pest in different countries, and the in vitro system of the insect could be a useful tool for isolation and characterization of the pathogens and physiological responses of the insect. In this context, a cell line was derived from the hemocytes of the European corn borer and was named AFKM-On-H for, respectively, O. nubilalis, Armand Frappier, King Mongkut Institutes, and Hemocytes. This cell line was initiated and maintained in Ex-Cell 400 medium supplemented with 10% heat-inactivated fetal bovine serum. The cells, mostly spherical in shape, not firmly attached to the plastic culture flasks, were passaged up to 200 times by repeated gentle pipetting of the cells. The doubling times at the 80th and 125th passages at 28°C and at the 122th and 169th passages at 25°C were 40, 29, 35, and 34 h, respectively. The AFKM-On-H cell line was further characterized by the morphology, karyotype, random amplified polymorphic DNA analysis, and isozyme profiles. Susceptibility of the cell line to cytoplasmic polyhedrosis viruses (CPV) Euxoa scandens (EsCPV), Dendrolimus punctatus (DpCPV), and Choristoneura fumiferana (CfCPV); nuclear polyhedrosis viruses [Autographa californica (AcMNPV) wild type and recombinant, Antherea yammamai (AnyaNPV)]; and Chilo iridescent virus was demonstrated. Relative sensitivities of the cell line to Bacillus thuringiensis and Metarhizium anisopliae toxins and effects of the molting hormone 20-hydroxyecdysone on this new hemocyte cell line were characterized.     

中华卵索线虫的体外培养

在研究中华卵索线虫的体外培养方法的同时,对其在不同培养基中的生长发育情况进行了观察。结果表明：以培养基TC－199加20％热灭活胎牛血清的培养效果较为理想,大多数线虫可存活3个月,最大虫体长55．1mm,宽204．13um,其发育程度大致与该种索线虫在宿主粘虫体内寄生8－9天的情况相近,培养期间观察到2次蜕皮;第一次蜕皮在卵内,第二次在培养6－8天之后,口针消失,虫体内滋养物体发育明显,尾部附器已经形成,没有观察到生殖原基的发育。     

Isolation of vegetable wasps and plant worms, Cordyceps nutans, from fruit-body tissue

We isolated Cordyceps nutans from the stipe and abdominal tissues of fruit bodies using a surface sterilization method. Hyphal growth was observed in inocula from both the stipe and abdominal tissue. Some strains from discharged ascospores were obtained and colony characteristics were compared to the strains isolated from the tissues. Colonies of isolates from ascospores grew quite slowly. Isolates of 43 from the 52 examined fruit bodies formed colonies similar to those from ascospores. To confirm the success of isolation, we analyzed by PCR-RFLP of the ITS regions of rDNA samples from fruit bodies, isolates from fruit bodies, and isolates from ascospores. All the isolates obtained from stipe and abdominal tissues presented identical patterns. In this study, we report the first successful isolation of C. nutans from fruit-body tissue using a surface sterilization method.