Gemmatalis Larvae Research Articles

Evaluation of Insecticidal Activity of Diterpenes and Lignans from Aristolochia malmeana against Anticarsia gemmatalis

The insecticidal activity of hexane extracts from the roots and leaves of Aristolochia malmeana was evaluated against Anticarsia gemmatalis larvae by topical application. Extract from the roots was the most active and caused 50% mortality in larvae at 308.4 microg/microL. From this extract, a clerodane diterpene, (-)-kolavenic acid, and three lignans, (-)-kusunokinin, (-)-hinokinin, and (8 S,8' R,9 S)-cubebin, were isolated by chromatography and partition procedures and then evaluated for their insecticidal activities either individually or in pairs. (-)-Kusunokinin showed higher activity against A. gemmatalis (LD10=9.3, LD50=230.1 microg/microL) than the crude extract, and its activity was dose-dependent, whereas the other constituents did not exhibit any significant activity. Together with (-)-kusunokinin and (-)-hinokinin, (-)-copalic acid, (-)-2-oxokolavenic acid, (-)- ent-6-beta-hydroxy-copalic acid, (8 R,8' R,9 R)- and (8 R,8' R,9 S)-cubebins, (-)-fargesin, and (-)-phillygenin were isolated from the hexane extract of the leaves. The compounds were identified on the basis of spectroscopic analysis.

An Anticarsia gemmatalis multiple nucleopolyhedrovirus mutant, vApAg, induces hemocytes apoptosis in vivo and displays reduced infectivity in larvae of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)

An Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) mutant, vApAg, induces apoptosis in a cell culture derived from Anticarsia gemmatalis (UFL-AG-286), reducing viral progeny. We have investigated apoptosis induction in vivo by vApAg in A. gemmatalis larvae and its correlation to infectivity reduction. LC 50, LD 50, LT 50 and the mean time to death of larvae were determined for vApAg and AgMNPV. Apoptosis was accessed for hemocytes of infected larvae using light and transmission electron microscopy. All types of hemocytes can be infected by vApAg. After 12 h post-infection (h p.i.), typical cellular modifications associated to nucleopolyhedrovirus infection were observed. Apoptosis becomes evident after 24 h p.i., and massive after 72 h p.i. Necrosis of infected cells was also observed. Despite cell death, hemocytes produced budded viruses and polyhedra. Pl and gh1-type hemocytes presented phagocytic activity. Agarose gel electrophoresis revealed fragmentation of hemocytes DNA at late times post-infection. The LC 50 and LD 50 were between five- and six-fold higher for vApAg. The LT 50 and the mean time to death were higher for vApAg in a same treatment or for a similar mortality induced by AgMNPV. These results show correlation of apoptosis and the reduced infectivity of vApAg in A. gemmatalis larvae.

Susceptibility/resistance of Anticarsia gemmatalis larvae to its nucleopolyhedrovirus ( AgMNPV): Structural study of the peritrophic membrane

This investigation compares the peritrophic membrane (PM) morphology along the midgut of susceptible (SL) and resistant (RL) Anticarsia gemmatalis larvae to the AgMNPV. The PM increased the thickness from the anterior to the posterior midgut region in both insects strain; however, the intensity of FITC-WGA reaction of the PM in the RL were greater than in SL. The PM in RL was ultrastructurally constituted by several layers of fibrous/vesicular materials in comparison with the few ones in SL. Our results showed that the structure of PM in the RL could be one of the resistance barriers to AgMNPV.

A Recombinant Truncated Cry1Ca Protein Is Toxic to Lepidopteran Insects and Forms Large Cuboidal Crystals in Insect Cells

A truncated version of the cry1Ca gene from Bacillus thuringiensis was introduced into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) under the control of two promoters. A recombinant virus (vSyncry1c) was isolated and used to infect insect cells in culture and insect larvae. Structural and ultrastructural analysis of insects infected with vSyncry1C showed the formation of large cuboidal crystals inside the cytoplasm of insect cells in culture and in insect cadavers late in infection. Infected insect cell extracts were analyzed by SDS-PAGE and Western blot and showed the presence of a 65-kDa polypeptide probably corresponding to the protease processed form of the toxin. Bioassays using purified recombinant toxin crystals showed a CL(50) of 19.49 ng/ml for 2(nd) instar A. gemmatalis larvae and 114.1 ng/ml for S. frugiperda.

Temperature-dependent development of the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson in Anticarsia gemmatalis (Hübner) larvae (Lepidoptera: Noctuidae)

The influence of temperature on the development rate of the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson in Anticarsia gemmatalis (Hübner) 3rd-instar larvae was studied under constant temperature regimes. A non linear model was used to describe temperature-dependent vegetative and reproductive development rates of the fungus. The temperature required to reach the highest vegetative development rate was estimated to be 25.5ºC while lower and upper developmental thresholds were 11ºC and 30ºC, respectively. The rate of development for conidia release was stable from 18ºC to 26ºC. The estimated lower and upper thresholds of vegetative development coincide with conditions during the natural epizootics in central Argentina. Non-linear effects of temperature on vegetative and reproductive development rates and probability distributions of N. rileyi under in vivo conditions are described.

In vivo apoptosis induction and reduction of infectivity by an Autographa californica multiple nucleopolyhedrovirus p35− recombinant in hemocytes from the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)

Baculoviruses have long been shown to regulate apoptosis in cultured insect cells. Recently, this phenomenon was also reported to occur in vivo, reinforcing the importance of apoptosis in insect immunity against viruses. The vP35del virus, an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) recombinant, was previously shown to induce apoptosis in Anticarsia gemmatalis cultured cells. In order to verify the AcMNPV interaction with hemocytes, apoptosis induction in vivo and its effects upon infectivity, we studied the course of intrahemocoelic infection of recombinant viruses (vHSGFP and vHSGFP/P35del) in A. gemmatalis larvae. Insect development and mortality were monitored and infection progress was followed by light, fluorescence and electron microscopy. For all doses tested, vHSGFP/P35del caused lower mortality than vHSGFP. Mortality of 95% occurred with a dose of 4 × 10 6 PFUs of vHSGFP, which was reduced to 60% for vHSGFP/P35del. GFP expression was first observed at 3 h p.i. for the two viruses, increasing for vHSGFP (40% at 120 h p.i.) and decreasing for vHSGFP/P35del (0% at 120 h p.i.). The virus vHSGFP/P35del induced apoptosis in hemocytes, with some budded virus being produced, and fragmented cells were observed between 24 and 72 h p.i. The recombinant vHSGFP induced typical wild-type cytopathic effects, with low production of occluded viruses until 120 h p.i. Plasmatocytes and granular hemocytes type 1 were the hemocytes most susceptible to both viruses. For these experimental conditions, we concluded that A. gemmatalis is a semi-permissive host to AcMNPV; moreover, apoptosis reduces AcMNPV infectivity and the p35 gene is essential for blocking apoptosis in this system.

Histopathology of Anticarsia gemmatalis Hübner (Lepidoptera; Noctuidae) treated with Nucleopolyhedrovirus and Bacillus thuringiensis serovar kurstaki

The Anticarsia gemmatalis is responsible for the use of chemical insecticides in the soybean culture, causing a significant increase in the costs of farming and a great unbalance in the ecosystem. The use of microbial agents, like Bacillus thuringiensis serovar kurstaki (Btk) and Anticarsia gemmatalis nucleopolyhedrovirus (AgNPV), they are an alternative to chemical control of the pest insects. In the interaction analysis of the entomopathogenic bacteria and virus it is considered important the in vitro action mode of these microbiology control agents. Therefore, the present study aims the histopathological analysis of the A. gemmatalis larvae digestive system after the interaction in vivo of the entomopathogenic Btk and AgNPV, represented the Dipel and Baculovirus anticarsia formulations, respectively. The evaluations were realized in larvae of 2nd instar, in which the mortality was evaluated daily, and a histopathology was done with collected larvae in time of 1, 3, 6, 12 and 24 hours after the treatments application. The results of the in vivo assays reveal that the treatment using the association of AgNPV-Btk (98.68% of mortality) was more efficient than using AgNPV isolatedly (81.28% of mortality), but the Btk when used isolatedly had a mortality of 100%. The treatments showed significant (P<0.05) differences between AgNPV and Btk, AgNPV and AgNPV/Btk. The histopathological analysis of the AgNPV and Btk in A. gemmatalis larvae suggests that the Dipel and Baculovirus anticarsia products were more efficient when they were used simultaneously, because the action of AgNPV was intensified when used in association with Btk, causing changes in the larvae midgut after 6 hours of treatments. When the entomopathogens were used isolating the gut cells alterations were observed only 12 hours after the treatments.

Pathology of Anticarsia gemmatalis larvae infected by two recombinant A. gemmatalis multicapsid nucleopolyhedroviruses

Light and stereomicroscopy examinations of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV)-infected insects were performed in order to follow infection in its host, A. gemmatalis. Fourth-instar A. gemmatalis larvae were infected by administration of occluded virus (polyhedra) from two recombinant AgMNPV viruses (vAgEGTΔ-lacZ or vAgGalA2) directly into the larvae foregut. The recombinant virus vAgEGTΔ-lacZ has the β-galactosidase gene ( lac-Z) of Escherichia coli under the control of a constitutive promoter ( hsp70 from Drosophila melanogaster). The vAgGalA2 virus has the reporter gene lac-Z under the control of the AgMNPV very late polyhedrin gene promoter. At different times post-infection (p.i.) the infected larvae were dissected, fixed, and the product of the expression of the lac-Z gene detected by incubating the insects in a buffer containing X-gal. This allowed us to follow the infection through the blue cells (due to the degradation of X-gal by the enzyme Lac-Z). Insect larvae inoculated with polyhedra from the recombinant viruses showed midgut cells to be infected first, followed by tracheal cells, hemolymph, fat body, Malpighian tubules and brain cells. The infection was similar for the two recombinant viruses, with blue cells appearing earlier in insects infected with the vAgEGTΔ-lacZ virus when compared to the vAgGalA2 virus.

Survivorship, larval development and pupal weight of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) feeding on potential leguminous host plants

In Brazil, the velvetbean caterpillar Anticarsia gemmatalis Hübner is multivoltine. Several overlapping generations occur with larvae feeding on alternate host plants in areas of warmer temperatures. In this study, the survivorship and development of A. gemmatalis larvae were evaluated on 17 species of cultivated and non-cultivated legumes in laboratory conditions. Larval survivorship on summer legumes varied from 91% on soybean to 5% on showy crotalaria and on velvetbean. On winter legumes, survivorship of A. gemmatalis larvae varied from 90% on white lupin to 25% on common vetch. All larvae fed on yellow lupin died during early development. On non-cultivated summer legumes, 70% of the larvae on Indigofera suffruticosa Millsp., and 55% on I. truxillensis H.B.K. and I. endecaphylla Jacq. reached the pupal stage. Based on survivorship, larval development and pupal weight, the most suitable legumes for A. gemmatalis were soybean, pigeon pea, and white lupin.

Molecular characterization of Bacillus thuringiensis strains from Argentina.

Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.

Morphological study of the hindgut in larvae of Anticarsia gemmatalis Hübner(Lepidoptera: Noctuidae)

The velvetbean caterpillar (Anticarsia gemmatalis Hubner) has great economical interest as it affects the soybean crop worldwide. This work describes the morphology of the hindgut in A. gemmatalis larvae emphasizing their histological aspects. Distinct morphological regions, identified as pylorus, ileum, colon and rectum, constitute the hindgut of A. gemmatalis. A thin cuticular intima, a simple epithelium and muscular layer compose the hindgut wall, independent of the region. Microspines project from the cuticular intima in the posterior interstitial ring, between the midgut and the hindgut, and the posterior pyloric region. A single circular layer of large fibers, differing from the other hindgut regions that present two layers of muscular fibers, forms the rectal musculature. The distal ends of Malpighian tubules cross the rectum wall and constitute the cryptonephric excretory system typical in Lepidoptera larvae.

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase ( egt ) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus ( egt −) AgMNPV and its virulence towards its insect host. We have inserted an hsp70 - lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ- lacZ ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ- lacZ compared to the wild-type virus. A mean of 20.4×10 8 OBs/g/larva and 40.7×10 8 OBs/g/larva was produced from vAgEGTΔ- lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC 50 ) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ- lacZ . The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ- lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.

Phylogenetic relationships among strains of the entomopathogenic fungus, Nomuraea rileyi, as revealed by partial beta-tubulin sequences and inter-simple sequence repeat (ISSR) analysis.

To elucidate the phyletic relationships among three members of the entomogenous fungal genus, Nomuraea, with an emphasis on N. rileyi. Relationships were evaluated by analysis of the beta-tubulin gene and of inter-simple sequence repeats (ISSR). The amplification product of the partial beta-tubulin gene was larger for N. atypicola than for N. rileyi, and sequencing of this gene fragment confirmed that N. atypicola possesses approximately 25 more nucleotides than N. rileyi and N. anemonoides. Based on neighbor joining and bootstrap analysis of the partial beta-tubulin gene, N. atypicola failed to form a monophyletic grouping with the other two species of Nomuraea. In contrast, the single isolate of N. anemonoides clustered with the N. rileyi isolates, and both taxa grouped with Epichloe typhina (Hypocreales: Clavicipitaceae). Results from this study suggested that N. rileyi and N. anemonoides are closely related to the Clavicipitaceae. In contrast, evidence indicated that N. atypicola is not closely related to this family, and that this taxon is not a Nomuraea. Based on the 83 polymorphic loci of ISSR, it was observed that isolates of N. rileyi from diverse geographical origins were distinctly different from both N. atypicola and N. anemonoides. Considerable heterogeneity was observed in the 18 isolates of N. rileyi tested, and several clusters contained isolates from disparate geographical locations and hosts. However, three isolates from the Philippines (three host species) and three strains isolated from velvetbean caterpillar (Anticarsia gemmatalis) larvae in South America did cluster together. Two other strains from Brazil (isolated from Spodoptera spp.) were distinct from the velvetbean caterpillar isolates from South America. The beta-tubulin gene was generally too conserved to resolve intraspecies variability. However, ISSR did identify polymorphisms among the isolates of N. rileyi tested. The results of this study indicate that ISSR may be used as robust molecular markers for studying the population genetics of this entomopathogenic fungus.

Stability and Persistence of Two Formulations Containing Anticarsia gemmatalis Nuclear Polyhedrovirus (AgMNPV)

The stability and the persistence of two AgMNPV formulations developed by the Instituto Biológico and the Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Brazil were studied. Formulations were stored in environmental conditions and exposed to ultraviolet radiation in laboratory and to solar radiation in the field. The wettable powder formulation (WP) was prepared by impregnating viral suspensions + adjuvants on inert mineral, chiefly kaolin. The emulsifiable oil formulation (EO) was prepared by mixing the pathogen suspension with corn oil + adjuvants. At 120-day intervals for 20 months, samples of formulations and the standard treatment (crude virus) kept in a laboratory cabinet were added to artificial diet fed to 4th-instar Anticarsia gemmatalis (Hübner) larvae. After 20 months in storage, the formulation EO lost only 18.3% of its original activity, whereas the formulation WP had its efficiency reduced to 11.7% after 12 months. No significant differences between the two formulations were observed when exposed to the ultraviolet germicidal light, and both formulations protected the virus when compared to the standard treatment. Under field conditions, the EO formulation enhanced the viral persistence, with >60% of the original activity present 14 days after the application. Most of the WP formulation was removed from leaves by the rain.

Detection of cry1 genes in Bacillus thuringiensis isolates from South of Brazil and activity against Aanticarsia gemmatalis (Lepidoptera:Noctuidae)

The bacterium Bacillus thuringiensis (Bt) is characterized by its ability to produce proteic crystalline inclusions during sporulation. Cry1 protein has insecticidal activity and is highly specific to certain insects and not toxic to unrelated insects, plants or vertebrates. In this work, the patogenicity of twelve Bt isolates was tested against Anticarsia gemmatalis, one of the most important insect pests of soybeans. Spore-crystal complex was applied to the surface of artificial diets and the mortality of A. gemmatalis larvae was assessed seven days after each treatment. When compared to a control Bt isolate known by its high toxicity to A. gemmatalis larvae, four novel Bt isolates exhibited even higher toxic activities against the insect, resulting in more than 90% mortality. PCR was used to amplify DNA fragments related to known cry1 genes. Bt strains with high toxicity produced expected PCR products of around 280 bp, whereas non-toxic or low toxic strains did not produce any PCR product or showed amplified fragments of different sizes. Toxic Bt isolates also exhibited an expected protein profile when total protein extracts were evaluated by SDS-PAGE.

Fluorescent Brighteners Improve Anticarsia gemmatalis (Lepidoptera: Noctuidae) Nucleopolyhedrovirus (AgMNPV) Activity on AgMNPV-Susceptible and Resistant Strains of the Insect

Abstract Fluorescent (optical) brighteners are known for their characteristics of protecting baculoviruses against deactivation by ultraviolet (UV) light and enhancing the activity of these agents as microbial insecticides on hosts and semipermissive hosts. These substances were evaluated in combination with the velvetbean caterpillar, Anticarsia gemmatalis Hubner, multiple-embedded nucleopolyhedrovirus (AgMNPV). The first trial involved 4 fluorescent brighteners (Blankophor BBH, Blankophor HRS, Blankophor RKH, and Tinopal LPW) obtained from the United States. The second trial was conducted with 11 fluorescent brighteners (Tinopal UNPA-GX, Tinopal DMS, Tinopal CBS, Leukophor DUB, Leukophor BSBB, Hostalux KS-N, Hostalux ETBN, BRY 10 D2 100, BRY 10 D2 150, Uvitex BHT, and Uvitex NFW) available in Brazil in combination with the AgMNPV to determine the degree of enhancement of viral activity. These brighteners were also evaluated with regard to AgMNPV protection against deactivation by UV light. Combinations of the virus with selected fluorescent brighteners were tested against both AgMNPV-susceptible and resistant strains of A. gemmatalis. In the first trial, brighteners obtained from the United States promoted increases in AgMNPV activity from 5.2-fold (Blankophor HRS) to 76.6-fold (Blankophor RKH) and reduced the mean time to death by 2.8 to 3.5 days. In the second trial, the most effective brightener (Tinopal UNPA-GX) reduced the LC50 in A. gemmatalis larvae from 7083 occlusion bodies (OBs)/ml (virus alone) to 77.8 OBs/ml (≈90-fold). When 4 selected brighteners were tested in combination with the AgMNPV in resistant insects, the LC50 was reduced by ca. 10,000-fold (Leukophor DUB) to ca. 62,000-fold (Tinopal UNPA-GX), in comparison to the LC50 of 3.7 × 107 OBs/ml observed for the virus alone. Therefore, mortality of highly resistant A. gemmatalis larvae to the AgMNPV increased dramatically when the virus was combined with some fluorescent brighteners. UV protection measured by original activity remaining (OAR) varied from 90% OAR (Tinopal UNPA-GX and BRY 10 D2 100). All efficacious brighteners were stilbene disulfonic acid derivatives and, when used alone, none showed negative effects against A. gemmatalis larvae.

Effects of ambient UV-B radiation on soybean crops: Impact on leaf herbivory by Anticarsia gemmatalis

Replicated field experiments with large plastic filters were carried outin Buenos Aires (Argentina, 34° S) to study the impacts of current levelsofsolar UV-B radiation (λ ≤ 315 ) on soybean(Glycine max L.) crops and their interactions with chewinginsects, in particular the soybean worm Anticarsiagemmatalis Hübner (Lepidoptera: Noctuidae). Solar(near-ambient)UV-B induced changes in the leaves that reduced their attractiveness toA. gemmatalis larvae in laboratory “choice”bioassays. When the A. gemmatalis larvae were forced toconsume leaves from field plots that received solar UV-B, they grew slightlyless rapidly and suffered more mortality than their counterparts fed withleavesfrom plots covered with polyester films that excluded the UV-B component ofsunlight. Exposure of the larvae themselves to ambient UV-B under a soybeancanopy during the feeding trials did not lower their life expectancy. At thewhole canopy level, we found that solar UV-B exclusion resulted in a two-foldincrease in the number of leaf lesions inflicted by various species of chewinginsects that naturally invaded the field plots. Leaves from canopies exposed tosolar UV-B showed significantly higher levels of soluble phenolics and lowerlevels of lignin than leaves that developed in canopies covered by polyesterfilms. No differences in specific leaf mass, leaf nitrogen or hemicellulosecontent were detected between the control and the solar-UV-B exclusiontreatments. Our results are consistent with the idea that present-day solar UV-B has an important regulatory influence on the interactions between plants and phytophagous insects.

Polymerase Chain Reaction Techniques to Detect Multiple-Nucleopolyhedrovirus in Anticarsia gemmatalis (Lepidoptera: Noctuidae) and Predator Populations in Soybean

The detection of species and strains of baculoviruses in natural field insect populations facilitates ecological studies, risk assessment evaluation and quality control programs where wild type and/or recombinant baculoviruses are used. The polymerase chain reaction (PCR) was evaluated as a detection tool for determining viral persistence and spread in Anticarsia gemmatalis Hubner and predatory hemipteran populations in fields of soybean, Glycine max (L.) Merril. Two isolates of the A. gemmatalis nucleopolyhedrovirus (AgMNPV-2D and AgMNPV-D7) were applied individually and mixed in replicated field plots. The insect populations were sampled 1, 10, 15, 30, and 45 d after viral application and taken to the laboratory for PCR analysis. PCR analysis targeted the coding region of the viral polyhedrin gene and the highly variable homologous region 4 (hr4 region). The size of the expected products was 575 base pairs (bp) for the polyhedrin-PCR, and 1,726 bp (AgMNPV-2D) and 1,345 bp (AgMNPV-D7) for the hr4 region. The virus was detected in A. gemmatalis larvae and predators collected on all sample dates by PCR amplification of the polyhedrin region. AgMNPV was detected in the host and predator populations 1–45 d after application in the field. Virus movement into the control plots indicated that AgMNPV dispersed at a rate of 2–3 m per day. PCR amplification of the hr4 region was effective in distinguishing between AgMNPV-2D and AgMNPV-D7 isolates. The polymerase chain reaction proved to be a useful tool in ecological studies of this baculovirus.

Distance and Rate of Spread of Anticarsia gemmatalis (Lepidoptera: Noctuidae) Nuclear Polyhedrosis Virus Released into Soybean

The nuclear polyhedrosis virus of Anticarsia gemmatalis (Hubner) was released into soybean fields at two sites in Louisiana. The spread of the virus outside release plots was monitored by sampling A. gemmatalis larvae, predatory arthropods and parasitoids of A. gemmatalis , and soil. According to A. gemmatalis sampling, the virus spread at a rate of 0.8 and 1.2 m/d at the Crowley and Baton Rouge sites, respectively, within ≍6 wk after it was released. The virus spread distances of up to 44 and 58 m at Crowley and Baton Rouge, respectively, during that season. The rate of spread detected in A. gemmatalis was significantly ( P > 0.01) correlated with the rate detected by sampling and bioassay of predatory arthropods and parasitoids, primarily the former. There also was circumstantial evidence implicating birds and farm machinery in viral transport; 80% ( n = 15) of bird droppings sampled at the Baton Rouge site during viral spread contained viable A. gemmatalis nuclear polyhedrosis virus. The rate of spread did not differ by direction from release plots. The prevalence of viral disease decreased at increasing distances from release plots for 3 yr after release at the Crowley site and during the 1st yr at Baton Rouge. The amount of virus in soil after the first season at the Crowley site decreased from 3,598 polyhedral inclusion bodies per gram in the release plots to 131 and <1 polyhedral inclusion bodies per gram at distances of 46 and 114 m, respectively, but this decrease was not significant.

Lack of Vertical Transmission in Anticarsia gemmatalis (Lepidoptera: Noctuidae) Nuclear Polyhedrosis Virus, a Pathogen Not Indigenous to Louisiana

Baseline data were collected on the natural occurrence of the nuclear polyhedrosis virus of Anticarsia gemmatalis (Hubner) in Louisiana and Texas in preparation for future attempts at introduction and establishment of the virus. The virus was not detected in A. gemmatalis larvae collected from 12 sites in Louisiana or 2 sites in Texas (total n = 3,391) on numerous sampling dates in 1989 and 1990. The only exception was a possible, light infection in one insect collected at a site that had been sprayed with A. gemmatalis nuclear polyhedrosis virus during 1979–1981. The virus was not detected in any of 24 soil samples collected from two sites in Louisiana in spite of detection of as few as 15.9 polyhedral inclusion bodies per g of soil in positive controls. Attempts were made in the laboratory to determine whether the viral infections could persist in adults and be transmitted to the progeny. The A. gemmatalis nuclear polyhedrosis virus was not so transmitted, even though Spodoptera frugiperda (J. E. Smith) adults, the positive control, transmitted their homologous nuclear polyhedrosis virus to 14.3% of their progeny. It is hypothesized that a lack of vertical transmission is the reason that the A. gemmatalis nuclear polyhedrosis virus apparently is not indigenous to the United States.