Transmission agents of the nuclear polyhedrosis virus ofNeodiprion sertifer [Hym.: Diprionidae]

Colonies ofNeodiprion sertifer (Geoffroy) larvae infected by nuclear polyhedrosis virus were visited by parasitoids, predators, and scavengers. Six transmission pathways were studied in detail. Viable virus polyhedra (PIB) were isolated from external surfaces of parasitic wasps and faeces of tachinid flies, coccinellid larvae, and scavenging flies. Faeces of the scavenging flies remained infectious 9 months on foliage in the field. A bird fed on earthworms from contaminated soil passed infectious faeces. Raindrops on foliage below diseased larval colonies contained more than 10⁸ PIB/ml. It was concluded that although there are numerous transmission pathways, only a few may be available at any one time. The stochastic nature of many pathways implies that the overall transmission rate is highly variable. These results are consistent with frequent observations of low transmission levels between larvae of the same generation.      

Resistance ofSpodoptera frugiperda [Lep.: Noctuidae] to a nuclear polyhedrosis virus in the field and laboratory

Median lethal doses (LD₅₀s) of nuclear polyhedrosis virus (NPV) were determined in neonatal offspring ofSpodoptera frugiperda (J. E. Smith) (Sf) larvae captured in southeastern Louisiana in 1981, 1982, and 1984. These LD₅₀s ranged from 1.8 to 16.3 polyhedral inclusion bodies (PIB)/insect. The LD₅₀s significantly (P<0.05) increased during the season of 1982 but had no pattern in 1981 or 1984. However, the Sf populations increased in heterogeneity of response to the NPV during all 3 years. The LD₅₀ increased from 4.1 to 18.7 PIB/insect in a Sf laboratory colony exposed to the NPV LD₈₀ for 7 generations, whereas in a control colony not exposed to NPV the LD₅₀ was 5.9 PIB/insect after 7 generations.     

Factors affecting pathogenicity of NPV preparations to the corn earworm,Heliothis armigera

Pathogenicity ofHeliothis nuclear polyhedrosis virus (HSNPV) to the corn earworm,Heliothis armigera, was studied using 3 different inoculative methods. The LD50 values of 4^th-instar larvae inoculated with corn-fed, diet-fed and inoculum-imbiding method were 1.85×10⁶, 2.55×10⁵ and 1,22×10³ PIBs/larva, respectively. The inoculum-imbiding is more sensitive and convenient for inoculatingH. armigera with HSNPV. The HSNPV product, Elcar^®, was highly pathogenic toH. armigera, the LD50 values of 2^nd-, 3^rd- and 4^th-instar larvae being 27, 83 and 1,221 PIBs/larva, respectively, as measured by the inoculum-imbiding method. The mortality of 4^th-instar larvae caused by HSNPV was increased, but the incubation period was shortened with higher incubation temperatures. However, the high temperature at 35°C caused a lower mortality, and a prolongation of the median lethal time (LT50). Stability and persistence of HSNPV preparations were better in January–February and April–May than in June–July and October–November periods when sprayed on corn silks under field conditions. The HSNPV was inactivated by weak alkaline dew (pH 8.1) collected from soybean leaves, but it remained active on those from corn, tomato and asparagus with pH 7.2–7.3. The artificial heavy rainfall of 242 mm/h for 30 min did not wash off HSNPV preparations sprayed on the corn silks.     

A new cell line from the embryonic tissue of Helicoverpa armigera HBN. (Lepidoptera: Noctuidae)

A new cell line from the embryonic tissue of Helicoverpa armigera was established and designated as NIV-HA-197. It was maintained in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line at passage 20 had a heterogeneous population of cells consisting of mainly epithelial-like cells (70%), followed by fibroblast-like (27%), and multinucleated giant (3%) cells. The chromosome number ranged from 45 to 185. The growth curve at passage 40 showed a fivefold increase in cell number with a population-doubling time of approximately 60 h. The cell line was found infected with the microsporidium Nosema heliothids at passage 9. Using the antiprotozoan drug Metrogyl 400 and simultaneous heat treatment, the parasite was removed from the culture. The cell line can be cryopreserved for 30 mo. The species specificity of the new cell line was determined by studying the isoenzyme profile of four enzymes, viz., lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and glucose 6-phosphate dehydrogenase, and by heteroduplex analysis. Heteroduplex analysis was used to analyze the mitochondrial 16S ribosomal ribonucleic acid gene sequences along with the host insect gene sequences, and 100% homology was obtained, confirming the conspecificity of the cell line. The cell line was found to be susceptible to the baculoviruses Autographa californica multiple nucleopolyhedrovirus, Spodoptera litura multiple nucleopolyhedrovirus, and H. armigera single nucleopolyhedrovirus (HaSNPV). More than 90% of the cells were infected by HaSNPV on the seventh post infection day (PID), and 28.8 x 10(6) NPV/ml was yielded on the 10th PID. The in vitro-grown HaSNPV caused 100% mortality, when fed to the second instar H. armigera larvae, in 6 d. Cessation of feeding was observed on the second PID.     

Effect of larval age and dosage of nuclear polyhedrosis virus on the susceptibility of diamondback moth, Plutella xylostella

A laboratory experiment was conducted to study the efficacy of nuclear polyhedrosis virus (NPV) with nine concentrations against all stadia of Plutella xylostella (L). The susceptibility of the larvae was correlated negatively with the period of development of the larvae and positively with the virus concentrations. The highest mortality of 84% was recorded in first stadium larvae compared to lowest mortality of 38% in fourth stadium larvae. The LC₅₀ was 5.5×10¹ and 4.0×10⁴ polyhedral inclusion bodies (PIB)/ml for first and fourth stadium larvae, respectively.     

Virus particle packaging in baculovirus and cytoplasmic polyhedrosis virus inclusion bodies

The structure of the inclusion bodies (IBs) of three multiply enveloped nuclear polyhedrosis viruses (MNPVs), one singly enveloped NPV (SNPV), two granulosis viruses (GVs) and one cytoplasmic polyhedrosis virus (CPV) were compared. A method was devised to calculate the numbers of virus particles and nucleocapsids in IBs using data from light microscopy and thin sections. The three MNPVs, from Agrotis segetum (English and Polish virus isolates) and Mamestra brassicae had similar concentrations of virus particles ranging from 17.3 to 19.6 per μm³ of IB. Plusia gamma SNPV had a higher density of 59.6 virus particles per μm³ of IB, which partly compensated for its having smaller IBs (mean volume 0.65 μm³) than the MNPVs (2.60–9.71 μm³). The English A. segetum MNPV isolate had the most nucleocapsids in each virus particle (mean, 4.04) and the largest IBs (mean volume, 9.71 μm³), giving 674 nucleocapsids per IB on average. The GVs, from A. segetum and Pieris brassicae, mainly contained one nucleocapsid per IB. P. gamma CPV IBs had a much higher density of virus particles than the baculoviruses (260 per μm³ compared with 17–60 per μm³). These data are discussed in relation to the biological properties of these viruses, and possible adaptational advantages of alternative IB designs are considered.     

Transmission of a nuclear polyhedrosis virus inHeliothis zea [Lep.: Noctuidae] larval populations on soybean

Transmission of a nuclear polyhedrosis virus (NPV) inHeliothis zea (Boddie) on soybean was examinated. Artificial infestations ofH. zea were established at densities of 6.5, 19.5 and 58.5 larvae/row-m. Additional larvae infected to die from NPV in the 2nd stage were released into subplots to simulate 5 and 25% mortality levels. Virus transmission from infected to noninfected larvae was correlated with the initial incidence of infection in the population but not the density of larvae/row-m. Deposition of virus on plants from cadavers of larvae that died of virus infection was correlated with the initial incidence of infection in the populations and the density of larvae/row-m. After pupation of larvae in the 1st population, noninfected larvae only were again released to examine transmission of viral inoculum remaining on plants and soil. The percent mortality of larvae collected from the 2nd release was low and did not differ significantly between treatments. The concentration of virus on foliage and in soil after the 2nd release was directly correlated with density of larvae/row-m but not the incidence of infection within the population in the 1st release. This material is based upon work supported in part by the U.S. Department of Agriculture under Agreement N^o 82 CRSR-2-1000.     

Inapparent infection ofDiatraea grandiosella byHeliothis virescens cytoplasmic polyhedrosis virus

The terminal stage of infection with cytoplasmic polyhedrosis viruses (CPVs) is formation of crystal-like inclusion bodies (polyhedra) in host insects. The degree of susceptibility of larvae to CPV, based on light microscopy and presence of polyhedra, varies with the host species.Heliothis virescens (F.) andSpodoptera exigua (Hübner) are highly susceptible to CPV. In CPV treatedDiatraea grandiosella (Dyar), polyhedra were absent in all 400 + insects examined with light and electron microscopy. However,H. virescens larvae became infected when fed haemolymph ofD. grandiosella larvae or pupae (36±10 days post treatment) developed from CPV-treated larvae. No difference in pathology was observed betweenH. virescens larvae infected with CPV polyhedra and haemolymph fromD. grandiosella. This study provides evidence thatD. grandiosella can serve as a symptomless (no occlusion bodies) carrier of a CPV which is fully expressed inH. virescens species. The observation is interesting because it reveals a potentially important aspect of the epizootiology of this insect virus.     

A new variant of Autographa californica nuclear polyhedrosis virus

A variant of the baculovirus, Autographa californica nuclear polyhedrosis virus, has been isolated in Idaho during an epizootic disease in a field population of A. californica. Genotypic characterization indicates that the virus is distinct from variants previously characterized. Analysis of five clones, derived by plaque purification in cell culture, indicates relative homogeneity of the original virus isolate. Further exploration of the factors involved in natural genetic variability of baculoviruses is appropriate.     

Nuclear polyhedrosis virus for control of Spodoptera exigua larvae on glasshouse crops

the efficacy of a nuclear polyhedrosis virus, SeNPV, for controlling larval populations of the beet armyworm, Spodoptera exigua (Noctuidae) was studied in chrysanthemum, gerbera, kalanchoë and tomato crops in glasshouses. Applications of 1×10⁸ PIBs/m² on the various crops resulted in 95–100% larval mortality. Virus applications caused comparable levels of mortality in populations of early and late instar larvae. Reduction in feeding damage to the crops, however, was more pronounced when the larvae were early instars at the time of virus application. Single applications with the virus resulted in higher levels of larval mortality as double applications with 0.1% methomyl and diflubenzuron.The virus appears to be a potential control agent for the beet armyworm in Dutch glasshouses.

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Résumé Les résultats d'applications d'un virus de la polyédrose nucléaire contre des populations de chenilles de Spodoptera exigua sont examinés dans des chrysanthèmes, gerberas, kalanchoës et tomates. Une dose de 1×10⁸ PIBs/m² résulte à 95–100% mortalité larvale dans tous les végétaux. L'effet sur chemilles de première et seconde stade larvale et sur chenilles de troisième et quatrième stade larvale était pareille. Malgré le niveau élevé du mortalité larvale les végéteaux étaient damagés sérieusement, particulièrement quand le virus était appliqué contre des chenilles de troisième et quatrième stade larvale.Comparée avec des pesticides chimiques, méthomyl et diflubenzuron, applications de virus résultent à un mortalité larvale plus haute. Le conclusion était que le virus est un candidat sérieus pour l'utilisation dans le lutte biologique contre les chenilles de S. exigua dans des serres en Pays Bas.

     

Dispersal of the nuclear polyhedrosis virus of Neodiprion sertifer from soil to pine foliage with dust

Inclusion bodies (PIB) of nuclear polyhedrosis viruses (NPV) remain infectious for long periods in forest soils, but their transfer from soil to foliage is not well understood. Two instances of dispersal of the NPV of Neodiprion sertifer (Fourcroy) (Hymenoptera: Diprionidae) by dust are reported. The distributioo of diseased larval colonies in a young Scots pine stand indicated that dust caused by logging during winter could have started an epizootic. At another site there was a higher frequency of infected colonies near a forest road than further into the stand. The dust transmission hypothesis was tested by bioassays of road surface material and of samples of airborne dust collected on membrane filters. Both soil and air samples contained viable PIBs. Thus, virus dispersal with dust provided a likely explanation for the observed distributions of diseased larval colonies.

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Zusammenfassung Mortalität von N.sertifer Larven durch das wirtsspezifische Kernpolyedervirus ist zwischen den Massenvermehrungen der Sägewespe selten. Eine ständige Infektionskette bleibt nicht erhalten. Das einzige bekannte Reservoir von Virusinoculum zwischen den Massenvermehrungen ist der Waldboden. Die Übertragung von Virus vom Boden auf Kiefernnadeln wird als seltenes Ereignis betrachtet. Sie dürfte häufiger sein, wenn die bodenbedeckende Vegetation gestört ist. Es wird von zwei Studien berichtet, in denen offenbar NPV in windverfrachtetem Staub die Krankheit bei Larven verursachte.In einem jungen Bestand schottischer Kiefer, in dem keine neueren Meldungen von Virusauftreten vorlagen, wurden Proben genommen. Die Proben stammten aus verschiedenen Distanzen von einem ausgewachsenen, im vorherigen Winter gefällten Bestand. Die Befallsintensität sank hier scharf ab mit zunehmender Distanz vom Rand.Ferner wurde der Krankheitsbefall in Larvenkolonien an kleinen Bäumen nahe einer schmalen Forst-strasse und an Bäumen im Bestandesinnern untersucht: In Strassennähe war der Befall höher. Luftmuster wurden nahe der Strasse durch Membranfiltern gesaugt und in einem Biotest auf Virus untersucht. Wenn die Strassenoberfläche ungestört war, wurde kein Virus festgestellt. Wurde Staub aufgewirbelt, konnten infektiöse PIBs gefunden werden. Material von der Strassenoberfläche hatte eine hohe Virusaktivität.Die Übertragung des Virus vom Boden auf Kiefernnadeln dürfte demnach wichtig sein, um Krankheitsausbrüche auszulösen. Die beschriebenen Störungen waren vom Menschen verursacht. Ähnliches könnte sich unter andern Umständen ereignen, z.B. bei Windfall oder bei der Aktivität grösserer Tiere.

     

Dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by the digestive fluid of Trichoplusia ni (Lepidoptera:Noctuidae) larvae

The dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus by digestive fluid collected from 5th stage Trichoplusia ni larvae was studied in vitro. Observations were made at timed intervals using phase contrast microscopy, and scanning and transmission electron microscopy. Dissolution occurred rapidly and in a detectable sequence. Under phase contrast, most polyhedra lost their refringence by 0.5 min. The polyhedra became rounded in appearance with small protuberances on the surface and Brownian movement was observed within. After 1 min, the envelope of most polyhedra had ruptured, releasing the enclosed virions. The protuberances were also observed under the scanning electron microscope after digestion for 0.5 min. Many shell fragments devoid of internal contents were seen after more lengthy digestion. Internal structural changes were revealed by electron microscopy. After 1 min of exposure, polyhedra were observed in all stages of dissolution. By 3 min, only virions, scattered about in heterogeneous material, could be distinguished.     

Comparison of wild type and genetically engineered nuclear polyhedrosis viruses of Autographa californica for mortality, virus replication and polyhedra production in Trichoplusia ni larvae

Virus replication and polyhedra production of two polyhedron-positive recombinant nuclear polyhedrosis viruses of Autographa californica, AcJHE.KK and AcAaIT which encode juvenile hormone esterase and scorpion toxin, respectively, were compared with those of a plaque purified wild-type nuclear polyhedrosis virus, AcMNPV-C6, in Trichoplusia ni larvae. Though average times required to kill the T. ni larvae increased with the age of the larvae, killing time by either recombinant virus was significantly shorter than that by wild-type virus. Killing time was reduced ca. 30% for AcAaIT-infected larvae and 5 to 8% for AcJHE.KK-infected larvae as compared to that for AcMNPV-C6-infected larvae. The average weight of larvae infected with AcAaIT was significantly lower than that of larvae infected with AcJHE.KK and AcMNPV-C6. The mean numbers of polyhedra produced in each larva inoculated with AcAaIT and AcJHE.KK were ca. 20% and 60%, respectively, of those for AcMNPV-C6. Total virus titers in AcMNPV-C6-infected larvae were significantly higher than those in AcJHE.KK- and AcAaIT-infected larvae until 2 days post infection.     

Secondary transmission of a nuclear polyhedrosis virus ofNeodiprion taedae linearis [Hym.: Diprionidae] between larval colonies on loblolly pine

Transmission of a nuclear polyhedrosis virus (NtNPV) of the loblolly pine sawfly,Neodiprion taedae linearis Ross was examined in larval populations on loblolly pine. NtNPV infection of larvae in one colony resulted in transmission to other colonies in the tree. Mortality in untreated colonies was highest when the treated and untreated colonies were located in the upper canopy and when small larvae were treated with virus (42.6–61.9%). Levels of mortality from NtNPV in the untreated colonies were low (2.8–25.0%) when the treated colony was located low in the canopy. Larvae from untreated colonies in the tree were usually large at death. Mortality in untreated larval colonies collected from nearby trees was much less (0.0–27.7%) than that in the trees containing the treated colony. Published with the approval of the Director, Arkansas Agricultural Experiment Station. Use of a trade name does not imply endorsement or guarantee of the product or the exclusion of other products of similar nature.     

Dosage-mortality and time-mortality responses of the armyworm Spodoptera exempta to a nuclear polyhedrosis virus

Third-instar Spodoptera exempta larvae were fed on young maize leaves treated with 20 μl of polyhedral inclusion body (PIB) suspension of concentrations that varied from 1.6 × 10² to 1.6 × 10⁹ PIBs/ml. Daily observations were kept on mortality rates. A probit analysis on the results gave an LD₅₀ value of 48.4 PIBs/larva (lower and upper fiducial limits 39.2 and 59.4 PIBs/larva, respectively), and an LT₅₀ that varied from 146.2 to 221.3 hr, depending on the dosage. LD and LT values obtained show the high pathogenicity of S. exempta nuclear polyhedrosis virus to its host.     

Feeding and dispersion of Spodoptera exigua larvae and its relevance for control with a nuclear polyhedrosis virus

On chrysanthemum crops the larvae of the beet armyworm Spodoptera exigua (Hübner) (Lep.: Noctuidae) moved upwards to the top of plants after hatching and predominantly fed on the upper foliage layers. On tomato, however, the larvae did not move upwards and mainly fed on the lower leaves. On chrysanthemums up to the fourth instar most feeding occurred at the underside of foliage while the upper-epidermis remained intact. Larvae dispersing from a single egg batch of 35 eggs damaged about 90 small and 50 tall chrysanthemum plants during their development. The successive larval stages contributed respectively 0.1, 0.4, 4, 20 and 75 percent to the total foliage consumption.The results indicated that the virus preferably should be applied to the lower leaves of tomatoes and chrysanthemums when young instars are present, but to the upper-middle and top leaves of chrysanthemums when the larvae are older than second instars.

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Résumé La consommation et la dispersion des chenilles de Spodoptera exigua sont examinées sur chrysanthèmes et tomates de serre. Les adults déposent les oeufs généralement au feuilles près du sol. Apres l'éclosion les chenilles graduellement se mouvent en haute dans les chrysanthèmes et consomment principalement les feuilles les plus hautes. Dans tomate, cependant, les chenilles s'alimentent principalement au feuilles près du sol.Pendant leur dévelopment les chenilles originaires d'une seule pond de 35 oeufs peuvent endommager environ go petits ou so hautes crysanthè mes. Jusqu'à la quatrième stade larvale les chenilles s'alimentent principalement avec le surface inférieure des feuilles, sans consommer l'épiderme supérieure.Les cinq stades larvales contribuent respectivement 0,1, 0,4, 4, 20 et 75% a la consommation totale des feuilles de chrysanthème.Les resultats des expériments sur la conduite larvale suggestent que les viruses de la polyédrose nucléaire doivent être appliqués préférablement sur la face inférieure des feuilles bas de chrysanthème et de tomate avant que les chenilles se développent a la troisième stade larvale. Quand les larves sur chrysanthèmes sont déja dans le troisième stade larvale, application du virus sur les feuilles plus haute probablement donnait des resultats optimal.

     

Mapping of BamHI and SmaI DNA restriction sites on the genome of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica

The DNA of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica (AcNPV), has been analyzed with restriction endonucleases BamHI and SmaI. The molecular weight of the BamHI fragments, SmaI fragments, and BamHI + SmaI fragments has been determined. The molecular weight of AcNPV DNA is calculated to be about 82 million. A presumptive physical map of the BamHI and SmaI restriction sites on the AcNPV genome has been constructed.     

Bioevaluation of Subabul (Leucaena leucocephala) proteinase inhibitors on Helicoverpa armigera

Helicoverpa armigera, a highly polyphagous pest, has a broad host spectrum, causes significant levels of yield loss in many agriculturally important crops. Serine primarily responsible for most of the proteolytic activity in the larval gut of lepidopteron insects. Neonate larvae were reared on artificial diet and chickpea seeds smeared with Subabul Trypsin Inhibitor. Larvae fed with artificial diet showed reduction in larval weight up to 21% (HSTI) and 43% (LSTI). However, larvae fed on seeds showed significant reduction in weight, 52.4% (HSTI) and 60.3% (LSTI), suggesting that the diet also plays a vital role on the effectiveness of the inhibitors on larval growth and development. HSTI and LSTI inhibited the gut proteinases from larvae fed on artificial diet significantly (41.40% and 64.36%) compared to the gut proteinases (27.80% and 38.90%) from larvae fed on chickpea seeds. Seeds smeared with 10,000 TIU resulted in complete mortality of larvae while there was no mortality observed in artificial diet. The results reveal that LSTI is a stronger inhibitor of insect gut proteinases and for larvae fed with poor nutrition in the natural ecosystems, low level expression of inhibitor would be enough to affect the growth and development. Handling editor: Chen-Zhu Wang