Changes observed in hemolymph proteins of the lawn armyworm,Spodoptera mauritia acronyctoides,during growth,development, and exposures to a nuclear polyhedrosis virus

Vertical electrophoresis with acrylamide gel was used to study the effects of a nuclear polyhedrosis virus (NPV) on the hemolymph proteins of Spodoptera mauritia acronyctoides. An electrophoretic pattern consisting of 20 basic bands of proteins was separated in hemolymph of normal larvae which were older than 17 days. These hemolymph proteins increased quantitatively during growth. All 20 proteins could not be detected in hemolymph of younger larvae by the techniques utilized. Additional proteins were separated with metamorphosis. Lethal doses of NPV resulted in a general reduction of hemolymph proteins (hypoproteinemia) in infected larvae. Sublethal doses of NPV elicited an increase in certain hemolymph proteins. Similar increases in proteins were also observed in larvae surviving ostensibly lethal levels of NPV, in larvae subjected to physical stress, and in larvae reared axenically without formaldehyde in their diets. These same proteins, however, were present in approximately the same quantities in mature larvae. Physiopathology of NPV in S. mauritia appears to involve stress factors, host reactions, and the host endocrine system.     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.     

Mode of transmission of nuclear-polyhedrosis virus to progeny of adult Heliothis zea

Late second-instar Heliothis armigera larvae were infected with a granulosis and a nuclear polyhedrosis virus, and all the externally visible symptoms for each virus are described. The effects of the virus infections on the feeding habits of the insects are also described, and it was found that a granulosis infection can prolong the larval period by up to 100%. The larvae continue feeding during this prolonged larval period, and can reach almost double the size and mass of normal larvae.It was further found that each of the viruses displays a distinct set of symptoms which could indicate beyond any doubt which of the two viruses induced death in the host.     

Evaluation of the single radial diffusion technique for detection of nuclear polyhedrosis virus (NPV) infection in Heliothis zea

The single radial diffusion test is an effective method for detection of nuclear polyhedrosis virus infection in Heliothis zea larvae. Virus antigens were detected in some instances 48 hr after the larvae were exposed to virus. Most larvae tested positively for virus antigens 72 hr after exposure to the virus. The tests could be read within 4 hr if the incubation temperature was 35°C, and within 24 hr at 22°C.     

High-efficient expression of porcine IL-2 with recombinant baculovirus infected silkworm,Bombyx mori

Biologically active porcine Interleukin-2(poIL-2) was produced fromin vitro andin vivo baculovirus expression systems, namely theAutographa californica nuclear polyhedrosis virus (AcNPV)-cell culture system and the Hybrid nuclear polyhedrosis virus (HyNPV)— silkworm larva system. The concentration of the recombinant poIL-2(rpoIL-2) in the larvae hemolymph was 1 to 3 mg/mL, which was about 7 to 20 times those of the cell culture systems. The level of this expression efficiency is equal to that with transgenic livestock, secretion products in milk.     

Hemolymph responses in Heliothis zea to inoculation with Bacillus thuringiensis or Micrococcus lysodeikticus

Direct injection into the hemolymph of Heliothis zea of either an entomopathogen (Bacillus thuringiensis subsp. kurstaki) or a nonpathogen (Micrococcus lysodeikticus) is followed by a rapid phagocytosis and extensive removal of the organisms within 2 hr. The bacteria that survive this initial clearance initiate a new round of growth that is clearly evident 6–8 hr after injection. When the infecting organism is M. lysodeikticus, a second period of clearance occurs 8–12 hr after injection and nearly complete removal (many by lysis) is evident by the 12th hr. Larvae usually survive infection with this organism. When B. thuringiensis is the infecting organism, 60–80% of the phagocytized bacteria are lysed, however, the second wave of clearance seen with M. lysodeikticus does not occur; instead, the bacteria multiply extensively and death of the larvae results 12–16 hr after injection. This death does not appear to be caused either by crystalline protein or by the β-exotoxin. Analysis of hemolymph proteins using one-dimensional polyacrylamide gel electrophoresis indicated that although some quantitative changes were observed in some experiments, in the faster moving proteins when the infecting agent was B. thuringiensis, they were not consistent enough to support the idea that hemolymph proteins were either synthesized or used up during the time larvae were responding to the infectious agent. Dramatic changes were evident when the larvae were near death. No changes were ever observed when M. lysodeikticus was used as the infecting organism. A rapid response to infection using free spores of B. thuringiensis (sickness within 2–4 hr followed by death at 6–8 hr) may indicate that the spore germinating process is accompanied by release of a highly toxic material.     

Effect of larval maturation on mortality induced by nuclear polyhedrosis and granulosis virus infections of Heliothis armigera

All the instars of Heliothis armigera larvae were found to be susceptible to both nuclear polyhedrosis virus (NPV) and granulosis virus (GV). An inverse relationship between mortality and larval age was found in the case of the NPV, while the GV displayed a rather erratic mortality pattern. A degree of maturation immunity against the NPV was found to exist, but the same is not true for the GV. The important role that pupation plays on the effect of a lethal infection is also discussed.     

Gut Bacteria Are Not Required for the Insecticidal Activity of Bacillus thuringiensis toward the Tobacco Hornworm,Manduca sexta

It was recently proposed that gut bacteria are required for the insecticidal activity of the Bacillus thuringiensis-based insecticide, DiPel, toward the lepidopterans Manduca sexta, Pieris rapae, Vanessa cardui, and Lymantria dispar. Using a similar methodology, it was found that gut bacteria were not required for the toxicity of DiPel or Cry1Ac or for the synergism of an otherwise sublethal concentration of Cry1Ac toward M. sexta. The toxicities of DiPel and of B. thuringiensis HD73 Cry⁻ spore/Cry1Ac synergism were attenuated by continuously exposing larvae to antibiotics before bioassays. Attenuation could be eliminated by exposing larvae to antibiotics only during the first instar without altering larval sterility. Prior antibiotic exposure did not attenuate Cry1Ac toxicity. The presence of enterococci in larval guts slowed mortality resulting from DiPel exposure and halved Cry1Ac toxicity but had little effect on B. thuringiensis HD73 Cry⁻ spore/Cry1Ac synergism. B. thuringiensis Cry⁻ cells killed larvae after intrahemocoelic inoculation of M. sexta, Galleria mellonella, and Spodoptera litura and grew rapidly in plasma from M. sexta, S. litura, and Tenebrio molitor. These findings suggest that gut bacteria are not required for B. thuringiensis insecticidal activity toward M. sexta but that B. thuringiensis lethality is reduced in larvae that are continuously exposed to antibiotics before bioassay.Bacillus thuringiensis has long been regarded as a bona fide entomopathogen that can produce an array of virulence factors including insecticidal parasporal crystal (Cry) toxins, vegetative insecticidal proteins, phospholipases, immune inhibitors, and antibiotics (31). B. thuringiensis establishes lethal infections in many insect species after intrahemocoelic inoculation (9, 10, 14, 26, 31), and the insecticidal activity of Cry toxins, which lyse the intestinal epithelium, can be synergized by the presence of viable B. thuringiensis spores (31). In each instance, synergism has been attributed to hemocoelic infection by B. thuringiensis.A novel hypothesis (6, 7) proposed that B. thuringiensis is incapable of killing Lymantria dispar, Manduca sexta, Pieris rapae, or Vanessa cardui in the absence of gut bacteria. Prior exposure of L. dispar larvae to a combination of four antibiotics severely reduced the subsequent toxicity of the B. thuringiensis-based (spores and Cry toxins) bioinsecticide, DiPel (Valent BioSciences) (6). Both larval susceptibility to B. thuringiensis and the number of culturable gut bacteria were found to be negatively correlated with the concentration of antibiotics to which larvae were previously exposed. Furthermore, a total reduction in larval susceptibility was coincident with the elimination of any detectable gut bacteria. Experimental reinfection with Enterobacter sp. strain NAB3, found in the guts of some populations of L. dispar larvae, was found to rescue the toxicity of B. thuringiensis, whereas reinfection with Enterococcus casseliflavus and Staphylococcus xylosus did not. It was also shown that while Escherichia coli, Enterobacter sp. strain NAB3, and B. thuringiensis could all grow in tryptic soy broth, B. thuringiensis alone could not grow in filter-sterilized plasma from L. dispar larvae. Finally, it was shown that the toxicity of Cry1Aa-expressing E. coli JM103 to L. dispar larvae was reduced by the prior exposure of larvae to antibiotics and could be eliminated when E. coli was also heat killed before use. It was concluded that B. thuringiensis-induced mortality results from a mixed infection of the hemocoel that must include bacteria capable of growth within the L. dispar larval hemolymph (6).Using the same methods, it was subsequently reported that prior exposure of Vanessa cardui, M. sexta, Pieris rapae, and Heliothis virescens larvae to antibiotics eliminated culturable bacteria and rendered larvae resistant to DiPel (7). Experimental reinfection of larvae with Enterobacter sp. strain NAB3 rescued DiPel toxicity in V. cardui, M. sexta, and P. rapae but not in H. virescens larvae. Using a continuous-exposure bioassay, the susceptibility of Pectinophora gossypiella to the Cry1Ac-based bioinsecticide MVPII was found to be increased by prior exposure to antibiotics. Toxicity from a 48-h exposure of L. dispar larvae to MVPII was reduced, but not eliminated, by prior antibiotic exposure and could be rescued by reinfection with Enterobacter sp. strain NAB3. It was concluded that “enteric bacteria have important roles in B. thuringiensis-induced killing of Lepidoptera across a range of taxonomy, feeding breadth, and relative susceptibility to B. thuringiensis” (7).The present work shows that gut bacteria are not required for the insecticidal activity of B. thuringiensis or Cry1Ac toxin toward M. sexta but that prior antibiotic exposure reduces larval susceptibility to B. thuringiensis.     

Susceptibility of Heliothis armiger to a commercial nuclear polyhedrosis virus

The susceptibility of Heliothis armiger larvae of different ages to a commercial nuclear polyhedrosis virus (NPV), Elcar, was determined by bioassay. The median lethal dosage (LD₅₀) increased 150-fold during the first week of larval life at 25°C, i.e., during development to early fourth instar, but daily feeding rate and thus potential virus acquisition also increased. A linear relationship was determined between log LD₅₀ and larval length, indicating that larval length constitutes a useful index for estimating the susceptibility of larval populations. Median lethal times (LT₅₀s) were similar for larvae tested at ages of 0 to 7 days and ranged from 3.6 to 8.0 days at 30°C. The amount of virus produced in a single, infected neonate was equivalent to 1.4 × 10⁶ LD₅₀s for neonates, a 900,000-fold increase on the dose supplied. The data support the practice of directing the NPV against neonates, but, on the basis of larval susceptibility alone, the age of larvae at treatment may not always be critical.     

The delta-endotoxin of Bacillus thuringiensis. V. On the occurrence of endotoxin fragments in hemolymph

Leucine-³H labeled crystals of Bacillus thuringiensis δ-endotoxin were fed to last-instar larvae of spruce budworm, Choristoneura fumiferana, eastern forest tent caterpillar, Malacosoma disstria, and silkworm, Bombyx mori. Radioactivity was detected in hemolymph 1 min after feeding in the first two species, but not until 3–5 min after feeding in silkworm larvae. Most of the radioactivity from hemolymph of all three species eluted from gel filtration columns at the same elution volume indicating similar moleculear weights (<1800 daltons).     

Activation of latent virus infections in larvae of Adoxophyes orana (Lepidoptera: Tortricidae) and Barathra brassicae (Lepidoptera: Noctuidae) by foreign polyhedra

The number of larvae containing polyhedra increased when larvae of Adoxophyes orana and Barathra brassicae were fed on polyhedra of nuclear polyhedrosis virus (NPV) of the reciprocal species. Comparison of restriction endonuclease EcoRI cleavage patterns of DNA isolated from polyhedra used as inocula and from polyhedra obtained after cross-inoculation showed that cross infection did not occur. The observations indicate that latent viruses were activated in both insects. Activation of the A. orana latent NPV with polyhedra of a cytoplasmic polyhedrosis virus (CPV) of B. brassicae, and cross-inoculation with an extract prepared from healthy larvae indicated that an activating agent does not have to be a component of nuclear polyhedra.     

Galleria mellonella infected with Bacillus thuringiensis involves Hsp90

Insects are good models for studying the innate immune response. We report that Galleria mellonella larvae infected with entomopathogenic bacteria Bacillus thuringiensis kurstaki show changes in the level of Hsp90. Our experimental approach was to pre-treat larvae with the Hsp90-binding compound, 17-DMAG, before infection with B. thuringiensis. We show that pre-treated animals display a higher level of immune response. This was mainly manifested by enhanced action of their hemolymph directed toward living bacteria as well as lysozyme activity digesting bacterial peptidoglycan. The observed phenomenon was due to the higher activity of antimicrobial peptides which, in contrast to healthy animals, was detected in the hemolymph of the immunestimulated larvae. Finally, the physiological significance of our observation was highlighted by the fact that G. mellonella pre-treated with 17-DMAG showed a prolonged survival rate after infection with B. thuringiensis than the control animals. Our report points to a role for Hsp90 in the immune response of G. mellonella after infection with B. thuringiensis at the optimal growth temperature.     

Influence of a baculovirus infection on molting and food consumption by Spodoptera litura

The effect of a baculovirus (nuclear polyhedrosis virus) infection on molting, neurosecretory activity, and food consumption of the tobacco leaf eating caterpillar. Spodoptera litura, is studied. The virus was administered by allowing the larvae to feed on castor leaves contaminated with purified polyhedral suspension (5.57 × 10⁸/ml). Host larvae failed to molt more than once after infection. Infected last-instar larvae failed to pupate and had prolonged larval stage. Activity of median neurosecretory cells (MNC) of brain increased during disease development. However, the larval—pupal molt was inhibited, possibly due to lack of release of neurosecretion from the MNC. The surface area of corpus allatum of last-instar larvae increased during infection indicating high activity. Infected larvae continued to feed almost until death. The indices of digestion and utilization were significantly lowered due to disease development.     

Effect of alkaline protease on the antigenic nature of wiseana nuclear polyhedrosis virus polyhedron protein

Polyhedron protein from Wiseana spp. nuclear polyhedrosis virus was found to be degraded by an alkali protease when polyhedra are dissolved in alkali. The protease activity did not occur at high pH (0.1 M NaOH) and was inactivated by heating polyhedra to 70°C for 3 h. The products from the protease degradation of Wiseana spp. nuclear polyhedrosis virus polyhedron protein retain the antigenicity of undegraded polyhedron protein as measured by the direct solid-phase radioimmunoassay and immunoadsorption. Degradation products below 27,000 daltons could not be detected by the sandwich radioimmunoassay, indicating that they are probably monovalent.     

Dynamics of Baculovirus Growth and Dispersal in Mamestra brassicae L. (LepidopteraNoctuidae) Larval Populations Introduced into Small Cabbage Plots

The nuclear polyhedrosis virus of Mamestra brassicae has been studied in larval populations of the moth introduced into small plots of cabbages. Primary dispersal of virus from single foci of infected larvae resulted from enhanced movement of the larvae, which colonized new plants logarithmically. Virus growth within the host population was quantified, and infection of young larvae in the following generation was related directly to the concentration of virus produced during the primary phase. Secondary cycling of virus resulted in dispersal of inoculum from multiple foci, and a large proportion of plants were ultimately colonized by infected larvae. The dynamics of virus growth during secondary dispersal were quantified and contrasted with results from the primary phase. The significance of these results is discussed in relation to possible control of insect pests through dispersal of virus by the host insect.     

Physiological and Nutritional Effects of a Fluorescent Brightener on Nuclear Polyhedrosis Virus-Infected Lymantria dispar (L.) Larvae (Lepidoptera: Lymantriidae)

Recent studies with gypsy moths (Lymantria dispar L.) show that the fluorescent brightener, Tinopal LPW, acts synergistically with the L. dispar nuclear polyhedrosis virus (LdMNPV), lowering the 50% lethality concentration of the virus (LC₅₀) by 1000-fold. As a continuation of this work, we investigated the effects of ingestion of LdMNPV/Tinopal on larval midgut pH, hemolymph pH, and nutritional indices. Larval feeding for 48 h on diet treated with either LdMNPV or Tinopal did not affect midgut luminal pH, which averaged 10.3. In contrast, after 48 h of larval feeding on LdMNPV/ Tinopal diet, midgut pH averaged 8.5, and frass production, weight gain, and larval nutritional indices were significantly reduced. These effects appeared to be irreversible after the first 24 h of feeding ad libitum on LdMNPV/Tinopal diet, and the reduction in midgut pH was not due to feeding cessation per se. No significant effects on hemolymph pH were detected with any treatment. No treatment effects were observed when "SITS," a related fluorescent brightener, was substituted for Tinopal. The severe biological perturbations resulting from LdMNPV/Tinopal consumption are interpreted in light of current understanding of larval lepidopteran midgut physiology.     

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.     

Juvenile hormone esterase is a biochemical anti-juvenile hormone agent

Juvenile hormone esterase, purified by affinity chromatography from the larval hemolymph of Manduca sexta in the fifth stadium, was injected into larvae of the same species in the earlier stadia resulting in a blackening of the cuticle following ecdysis to the next larval stadium. This anti-juvenile hormone response was dose-dependent for an injection in the second, third or fourth stadium. Cuticular blackening was prevented by treating larvae with the juvenoid epofenonane. Larval response to injected juvenile hormone esterase also varied with the time of injection within a single stadium, having a maximum effect for injections at the time of head capsule slippage. Juvenile hormone esterase activity measured from the hemolymph after injection of larvae in the second stadium decreased over an 11 h time-course. Because the anti-juvenile hormone effects resulting from a single injection of juvenile hormone esterase were dependent on the time of injection, it appears that when juvenile hormone biosynthesis is active in the insect, the duration of enzyme activity limits the anti-juvenile effects that can be induced.