Systematic differences in the population density of green spruce aphid, Elatobium abietinum in a provenance trial of Sitka spruce, Picea sitchensis

Quantitative bioassay techniques were used to measure the susceptibility of Heliothis armigera to three nuclear polyhedrosis viruses (NPVs): H. armigera singly-enveloped NPV (HaSNPV), H. zea SNPV (HzSNPV) and H. armigera multiply-enveloped NPV (HaMNPV). Viruses were identified by EcoRI restriction endonuclease analysis. Electrophoretic profiles of DNA fragments revealed that the HaSNPV isolate was a previously undescribed genotypic variant. Bioassays with neonate and 6-day-old larvae measured small but significant differences in virulence between the three viruses. HzSNPV was the most virulent for neonate larvae with a median lethal dose (LD₅₀) of five polyhedra. HaMNPV was least virulent for 6-day-old larvae, with a LD₅₀ of 1400 polyhedra compared with 640–670 polyhedra for HaSNPV and HzSNPV. In addition, the median lethal time (LT₅₀) for infection with HaMNPV in neonate larvae was approximately 1·7 days longer than for the other viruses. Although they varied in virulence, each of the three viruses was sufficiently virulent to have considerable potential as a microbial control agent of H. armigera.     

Relative pathogenicity of nuclear polyhedrosis viruses from Heliothis punctigera and Heliothis zea for larvae of Heliothis armigera and Heliothis punctigera

Laboratory bioassays indicated that the potency of a nuclear polyhedrosis virus from Heliothis zea derived from a commercial American formulation was similar to that of a naturally occurring nuclear polyhedrosis virus from H. punctigera in Australia. Both viruses exhibited high virulence for neonate larvae of H. armigera and H. punctigera, the major pest species in this genus in Australia. Hence evaluation of the virus in Australia can proceed employing virus from either H. punctigera or H. zea.     

Role of the exoprotease InA in the pathogenicity of Bacillus thuringiensis in pupae of Hyalophora cecropia

Two geographical biotypes of Nomuraea rileyi (from Ecuador and the United States) were topically bioassayed against seven lepidopteran species, i.e., Anticarsia gemmatalis, Heliothis zea, Heliothis virescens, Heliothis subflexa, Pseudoplusia includens, Spodoptera exigua, and Trichoplusia ni. There was an average difference of 1.7-fold in mortality in how cultures of the same insect species from different sources responded to topical applications of either biotype of N. rileyi. Regression equations and LC₅₀ values were obtained for each insect species and fungal biotype combination. Larvae of S. exigua were equally susceptible to both biotypes of N. rileyi. Although larvae of A. gemmatalis were moderately susceptible to the Ecuadoran biotype, they were relatively nonsusceptible to the Mississippian biotype. Species of Heliothis (H. zea, H. virescens, and H. subflexa) were about equally susceptible to the Mississippian biotype. Larvae of H. subflexa and H. virescens, however, were significantly less susceptible than H. zea to the Ecuadoran biotype. When the integumental barrier was breached via intrahemocoelic injections, larvae of H. virescens were as susceptible as H. zea larvae to blastospores of either biotype of N. rileyi.     

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC₅₀s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.     

Replication and infectivity of the single-embedded nuclear polyhedrosis virus, Baculovirus heliothis, in homologous cell lines

Three cell lines of Heliothis zea and one cell line of Heliothis virescens replicated the singleembedded, nuclear polyhedrosis virus (NPV) of H. Zea, (i.e., Baculovirus heliothis) with concomitant production of polyhedral inclusion bodies (PIB). Between 20 and 60% of the H. zea cells produced PIB, whereas only 3% of H. virescens cells were found to produce PIB. The H. zea cell lines produced 10 to 20 times more PIB than did the H. virescens cell line. The PIB from all cell lines produced typical symptoms of an NPV infection when bioassayed against larvae of H. zea. More than 99% of the total viral activity of the final whole culture was due to the PIB.     

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 high incidence of parasitism ofHeliothis SPP. [Lep.: Noctuidae] larvae in cotton in Southeastern Arkansas

During 1981 and 1982, bollworm,Heliothis zea (Boddie), and tobacco budworm,H. virescens (F.), larvae (n=3,666) were collected from 41 cotton fields near Portland, Arkansas (USA) to assess the occurrence of parasitism. Three strategies were employed to controlHeliothis spp. in these fields: (1) release ofTrichogramma pretiosum Riley; (2) insecticidal control; or (3) inaction (check). Insecticide use in nonchemical control fields was reduced, but not eliminated.Heliothis spp. larvae collected in cotton had higher parasitism rates in 1981 (30.9%) and 1982 (50.1%) than had been reported for cotton since the advent of organochlorine insecticide usage. Four species of larval parasites and 1 species of larval-pupal parasite were recorded. The larval parasiteMicroplitis croceipes (Cresson) comprised 90.6% and 94.5% of all parasitic insects reared from field collectedHeliothis spp. in 1981 and 1982, respectively. No difference (P>0.05) in level of parasitism existed betweenH. zea andH. virescens. Differences between treatments occurred only in 1982 whenH. zea larvae were parasitized at a greater (P<0.05) rate in check fields (68.3%) than in insecticidal control fields (44.3%). Higher levels of larval parasitism in cotton fields may be a consequence of reduced insecticide usage and changes in materials applied, particularly the pyrethroids. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Dept. of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Yield and activity of theHeliothis zea single nuclear polyhedrosis virus propagated in cloned and uncloned lines ofHeliothis cells

Summary Heliothis cell lines originated from different laboratories were characterized by isoenzyme analysis and then evaluated for their ability to produce the single nuclear polyhedrosis virus ofHeliothis zea (HzSNPV). A cloned cell line (designated Hzlb3), whose homogeneity was supported by both morphological and isoenzyme analysis, was derived from a parental line (Hzl). Significantly greater yields (about 10-fold) of tissue-culture-derived, non-occluded virus (TCNOV) were obtained when compared to the parental line. The Hzlb3 clone also gave significantly higher yields of TCNOV than the Hz3 and UND-K cell lines. Although lines Hzl, Hz3, and Hzlb3 produced significantly more polyhedral inclusion bodies (PIB) than line UND-K, the infectivity of PIB from UND-K equaled that of lines Hzl and Hzlb3.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Age-specific survivorship analysis ofHeliothis spp. Populations on cotton

Population dynamics of Heliothis virescens (F.) and Heliothis zea (Boddie ) (Lepidoptera: Noctuidae) eggs and larvae were studied for two years in a small plot of cotton, Gossypium hirsutum (L.). Due to morphological and ecological similarities, the pooled Heliothis population was considered for most of the analyses. Two generations of Heliothis eggs and larvae were completed during each year. Stage recruitment was estimated for the eggs and larval instars 2–6, and recruitment variances were estimated by a Monte Carlo method. A modified form of the Weibull distribution was developed and used as a model to characterize survivorship curves for each of the four Heliothis generations. A Type I survivorship curve (mortality rate increasing with age) was inferred for both Generation 1 (early season) data sets, whereas a Type II survivorship curve (mortality rate constant and thus independent of age) was inferred for both Generation 2 (late season) data sets. The shapes of the survivorship curves for the individual H. virescens and H. zea populations were inferred to be the same as those for the pooled populations. Analysis of the contributions of various factors to Heliothis stage-specific mortality indicated that natural enemies (predators and parasites) and the availability of food for larvae were responsible for between-generation differences in survivorship patterns.     

Characterization of clonal populations of theHeliothis zea cell line IPLB-HZ 1075

Summary A total of 24 clones (HZ 1075/UND-A through X) were initially isolated by dilution plating from the established IPLB-HZ 1075 cell line. Many of the isolates with highly vacuolated cytoplasms eventually died during subculturing. The cloned cell strains differed in their predominant morphology, cell doubling times, and relative ability to support replication of the singly encapsulated nuclear polyhedrosis virus ofHeliothis zea (HzSNPV). The origin of the cloned cell strains was confirmed by comparing their isozyme profiles with those of the parental IPLB-HZ 1075 cell line andH. zea larvae using stains for fumerate hydratase, lactate dehydrogenase (LDH), and malic dehydrogenase (MDH). One dipteran and several lepidopteran cell lines maintained in our lab were also separable using stains for LDH and MDH. This research was supported in part by USDA grant number GAM 8400211, and by a Jesse Jones Faculty Research Award from the University of Notre Dame to M. J. F.     

Electron microscopical studies and restriction analysis ofHelicoverpa armigera nucleo polyhedrosis virus

Nuclear polyhedrosis virus ofHelicoverpa armigera (HaNPV) from Madurai (south India) was isolated, purified and subjected to electron microscopical studies. The results indicate that the isolate is a multi embedded form of NPV. Further, STEM observations on polyhedra and polyhedral envelope of the HaMNPV are presented. The restriction pattern and genome size of HaMNPV Madurai isolate are compared with HaNPV isolates reported earlier. A dendrogram is presented to show the relatedness among the isolates.     

The infectivity of a nuclear polyhedrosis virus of the velvetbean caterpillar for eight noctuid hosts

Eight species of noctuid larvae were tested for susceptibility to a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis. Velvetbean caterpillar larvae were highly susceptible to crude preparations of polyhedral inclusion bodies (PIBs; LD₅₀ = 4.7 PIBs/larva), but preparations of purified polyhedra were much less effective against these larvae (LD₅₀ = 319.7 PIBs/larva). Of seven other noctuid species tested, only Heliothis virescens was as susceptible to the virus as A. gemmatalis. High dosages were required to kill Heliothis zea, Trichoplusia ni, Pseudoplusia includens, and Spodoptera ornithogalli. Plathypena scabra and Spodoptera frugiperda were not susceptible.     

Populations of Predaceous Natural Enemies Developing on Insect-Resistant and Susceptible Tomato in North Carolina

Naturally occurring populations of immature and adultGeocoris punctipes,adultColeomegilla maculataand immature coccinellids were monitored on field-grown tomato lines susceptible and resistant toManduca sextaandHelicoverpa zea. Helicoverpa zeaandHeliothis virescenseggs and small larvae that serve as prey for these predators also were monitored. MoreH. zeaandH. virescenseggs and small larvae were found on resistant than on susceptible plant lines. However, similar populations of largeH. zeaandH. virescenslarvae were found on resistant and susceptible plants. The number of adultGeocoris punctipes,adultColeomegilla maculataand immature coccinellids on resistant plants was always as high or higher than the number on susceptible plants. The data demonstrate no incompatibility of host-plant resistance with biological control provided by these predaceous insects, but indicate that the number ofG. punctipesand coccinellids required to provide effective biological control may develop too late in the season to be of practical value. Large populations of stilt bugs (Jalysus wickhami,Hemiptera: Berytidae) and spiders were observed to occur earlier in the growing season than eitherG. punctipesor coccinellids and may be a significant source of mortality forH. zeaeggs and small larvae.     

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.     

Susceptibility of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) to Vip3A insecticidal protein expressed in VipCot™ cotton

Susceptibility of laboratory and field colonies of Helicoverpa zea (Boddie) and Heliothis virescens F. to Vip3A insecticidal protein was studied in diet incorporation and diet overlay assays from 2004 to 2008. Responses of field populations were compared to paired responses of University of Arkansas laboratory susceptible H. zea (LabZA) and H. virescens (LabVR) colonies. After 7 d of exposure, observations were made on number of dead larvae (M) and the number of larvae alive but remaining as first instars (L1). Regression estimates using M (LC₅₀) and M plus L1 (MIC₅₀) data were developed for laboratory and field populations. Susceptibility of laboratory and field populations exposed to Vip3A varied among different batches of protein used over the study period. Within the same batch of Vip3A protein, susceptibilities of laboratory colonies of both species (LabZA and LabVR) were similar. Field colonies were significantly more susceptible to Vip3A than the respective reference colonies of both species. Within field populations, susceptibility to Vip3A varied up to 75-fold in H. zea and 132-fold in H. virescens in LC₅₀ estimates. Variabilities in MIC₅₀s were up to 59- and 11-fold for H. zea and H. virescens, respectively.     

Midgut pH in different instars of three Aedes mosquito species and the relation between pH and susceptibility of larvae to a nuclear polyhedrosis virus

Midgut pH values were determined for second, third, and fourth instar larvae of Aedes epactius, A. atropalpus, and A. scutellaris. Larvae were fed mixtures of pH indicators and kaolin clay and observed with a stereoscope to determine midgut pH. No significant difference was found in midgut pH values among the three species when the same instar was compared. However, significant differences were found in the length of the high (9.0 to 10.0) pH region when comparing the three instars for each species. Increasing age of treated A. epactius larvae caused a decrease in susceptibility to a nuclear polyhedrosis virus—from 56% infection for 12-hr-old larvae declining to 8% infection for 72-hr larvae. The increased length of the high pH region may relate to decreasing susceptibility of A. epactius to a nuclear polyhedrosis virus as the larvae age.     

A host-plant specialist, Helicoverpa assulta, is more tolerant to capsaicin from Capsicum annuum than other noctuid species

Plant secondary compounds not only play an important role in plant defense, but have been a driving force for host adaptation by herbivores. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), an alkaloid found in the fruit of Capsicum spp. (Solanaceae), is responsible for the pungency of hot pepper fruits and is unique to the genus. The oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae), is a specialist herbivore feeding on solanaceous plants including Capsicum annuum, and is one of a very few insect herbivores worldwide capable of feeding on hot pepper fruits. To determine whether this is due in part to an increased physiological tolerance of capsaicin, we compared H. assulta with another specialist on Solanaceae, Heliothis subflexa, and four generalist species, Spodoptera frugiperda, Heliothis virescens, Helicoverpa armigera, and Helicoverpa zea, all belonging to the family Noctuidae. When larvae were fed capsaicin-spiked artificial diet for the entire larval period, larval mortality increased in H. subflexa and H. zea but decreased in H. assulta. Larval growth decreased on the capsaicin-spiked diet in four of the species, was unaffected in H. armigera and increased in H. assulta. Food consumption and utilization experiments showed that capsaicin decreased relative consumption rate (RCR), relative growth rate (RGR) and approximate digestibility (AD) in H. zea, and increased AD and the efficiency of conversion of ingested food (ECI) in H. armigera; whereas it did not significantly change any of these nutritional indices in H. assulta. The acute toxicity of capsaicin measured by injection into early fifth instar larvae was less in H. assulta than in H. armigera and H. zea. Injection of high concentrations produced abdominal paralysis and self-cannibalism. Injection of sub-lethal doses of capsaicin resulted in reduced pupal weights in H. armigera and H. zea, but not in H. assulta. The results indicate that H. assulta is more tolerant to capsaicin than the other insects tested, suggesting that this has facilitated expansion of its host range within Solanaceae to Capsicum after introduction of the latter to the Old World about 500 years ago. The increased larval survival and growth due to chronic dietary exposure to capsaicin suggests further adaptation of H. assulta to that compound, the mechanisms of which remain to be investigated.     

Characterization and culture of virus replicating continuous insect cell lines from the bollworm,Heliothis zea (boddie)

Summary A series of five discrete virus replicating insect cell lines were isolated from the ovarian and fat body tissues ofHeliothis zea pupae. Two of these cell lines (IPLB-HZ-1075 and-HZ-1079) were studied in depth as to their growth and virus replication responses to specific nutrients (acetyl-β-methylcholine, fresh glutamine) in a number of media. The same two cell lines were identified to species by serological (microimmunodiffusion) and isozyme (phosphoglucoisomerase and peptidase:glycyl-leucine) techniques. Distinguishing comparisons were made with other cell lines that have been confused with the present lines in the literature and with cell line and host pupal extracts from the same and other lepidopteran species studied concurrently in this laboratory. Sterility culture tests were negative for mycoplasmas. The present fiveH. zea lines were the first insect cell lines to replicate polyhedra from a unicapsid multiple embedded nuclear polyhedrosis virus (Baculovirus Group A), in this case the homologous virus obtained from larvae ofH. zea.     

Alkanes of four related moth species,Helicoverpa and Heliothis

Comparison of the presence and quantities of cuticular hydrocarbons has been used successfully for identifying sibling species and races of several groups of insects. This approach has been extended to four species of moths previously regarded as belonging to the same genus, Heliothis. Gas chromatography was used to quantify the numerous high-molecular weight alkanes found on the cuticle of two pairs of closely related species: Helicoverpa zea and Helicoverpa armigera, and Heliothis virescens and Heliothis subflexa. Both sexes of H. zea and H. armigera contained different quantities of several alkanes that could be used for unambiguous identification. Similar comparisons of H. subflexa and H. virescens showed four peak ratios that were different for each species. Sexual dimorphism was minor in H. subflexa and H. virescens.