Histopathology of a nuclear polyhedrosis infection in Aedes epactius with observations in four additional mosquito species

Aedes epactius larvae were utilized to study the infection sequence of the nuclear polyhedrosis virus (NPV) from Aedes sollicitans. From 30 min to 6 hr postinoculation, polyhedra and many free virions were observed in the larval midgut lumen. Penetration of the midgut cells by virions was not observed. The first infected nuclei were observed 12 hr postinoculation. Nucleocapsids initially exhibited electron translucent cores which became electron dense before the nucleocapsids acquired an envelope. Envelope acquisition occurred through a process of de novo membrane morphogenesis. Occlusion of the singly embedded virions began by 18 hr postinoculation with the mature rough-surfaced polyhedra averaging approximately 1 by 2 μm. Unusually long nucleocapsids (approximately two or three times the length of other nucleocapsids) were only observed in late infection period nuclei. There was no evidence that long nucleocapsids represented an early developmental stage for nucleocapsids of standard length. Infection was restricted to midgut nuclei and gastric caecae cells. Infected early instar A. epactius larvae became moribund 36 to 40 hr postinoculation and infected midgut nuclei were observed to undergo lysis. The late stages of NPV infection were observed in larvae of A. annandalei, Wyeomyia smithii, Toxorhynchites brevipalpus, and Eretmapodites quinquevittatus. Virion development and occlusion in these species was basically identical to the sequence observed in A. epactius larvae.     

Susceptibility of larvae ofHeliothis zea,H. virescens,andH. armigera [lep.: Noctuidae] to 3 baculoviruses

The pattern of virulence (based on inclusion bodies) for 3 baculoviruses ofHeliothis, i.e. a unicapsid, nuclear polyhedrosis virus (HzSNPV); a multicapsid, nuclear polyhedrosis virus (HaMNPV); and a granulosis virus (HaGIV) was the same (HzSNPV>HaMNPV>HaGIV) for 3 species ofHeliothis. Based on numbers of nucleocapsids, however, the HaGIV was ca 2X more virulent than the HaMNPV for larvae ofH. virescens, (F.), and the HaMNPV was about 6X more virulent than the HaGIV for larvae ofH. armigera (Hübner). The fastest rate of larval mortality was obtained with HzSNPV. Although the mortality rate for HaGIV was faster than that of HaMNPV forH. virescens andH. armigera, it was slower than that of HaMNPV for larvae ofH. zea (Boddie). The pattern of susceptibility ofHeliothis species to HzSNPV and HaMNPV wasH. zea>H. virescens>H. armigera. Differences in susceptibility of the least susceptible species (H. armigera) and the most susceptible species (H. zea) to HzSNPV was ca. 1.6 X. Larvae ofH. zea, however, were ca. 4 to 6 X more susceptible to HaMNPV than were larvae ofH. virescens orH. armigera. A different pattern of susceptibility was recorded for HaGIV when larvae were challenged with HzSNPV and HaMNPV. Larvae ofH. virescens were ca. 20 and 35 X more susceptible to HaGIV than were larvae ofH. zea andH. armigera, respectively.     

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.     

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.     

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.     

The effect of soil pH on the persistence of cabbage looper nuclear polyhedrosis virus in soil

The persistence of singly embedded Trichoplusia ni nuclear polyhedrosis virus in Norfolk A loamy sand of various pH was studied under laboratory conditions. Virus extracts of the treated soils were bioassayed at three monthly intervals and showed that virus residues are affected by soil pH. Within the range of soil pH tested (4.83-7.17), the lower the pH, the more rapidly the virus was inactivated. The practice of liming fields for maintaining favorable physiological characteristics as well as for nutritional reasons, enhances the persistence of the virus in the soil.     

Acid and alkaline phosphatase activity in the fat body and midgut of the beet armyworm,Spodoptera exigua (Lepidoptera: Noctuidae), infected with nuclear polyhedrosis virus

Changes in the activity of acid and alkaline phosphatase in Spodoptera exigua larvae infected with nuclear polyhedrosis virus have been investigated. Three days after per os infection, the activity of acid phosphatase in the fat body and midgut of infected larvae was significantly higher than that in normal larvae. Alkaline phosphatase activity did not show such significant changes. There were differences in the phosphatase patterns depending on whether their activities were expressed as enzyme units per milligram of fresh organ weight or per milligram of homogenate protein. The literature relevant to the subject allows us to conclude that the increase in phosphatase activities in S. exigua larvae is not specifically associated with virus infection itself, but, rather, is a reaction of the insect organism to the diminishing supply of energy sources.     

Lack of absorption and rapid elimination of Rifampicin as factors precluding tests against nuclear polyhedrosis in Spodoptera littoralis larvae

Rifampicin administered per os in artificial medium to first-instar larvae of Spodoptera littoralis together with nuclear polyhedrosis virus did not affect virus infection and resulted in total larval mortality. A pharmacokinetic study of this antibiotic was carried out by means of a microbiological assay. Using Bacillus thuringiensis var. thuringiensis as the test organism for assaying the antibiotic, as little as 0.5 μg of Rifampicin could be detected in the presence of hemolymph, and no such detectable amounts could be found in the hemolymph of sixth instar larvae which had been fed the antibiotic. Most if not all of the antibiotic was in the feces, excreted in active form. When given parenterally, Rifampicin was detected in the hemolymph 30 min after injection but not 24 hr thereafter, when active antibiotic was found concentrated in the first fecal pellets evacuated after injection. This disappearance is ascribed to excretion via the Malpighian tubules. The larval responses detailed above preclude the testing of Rifampicin as an antiviral drug against nuclear polyhedrosis.     

Isolation and characterization of a synergistic enzyme from the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor than enhances the infection of a nuclear polyhedrosis virus in the armyworm, Pseudaletia unipuncta, was isolated from the occlusion body (capsule) of a granulosis virus of the armyworm. Disc electrophoresis indicated that the purified factor was a single homogeneous compound. Chemical identification and amino acid analysis showed that it was a simple protein, with a molecular weight of 152,000–163,000. Proteolytic enzymes did not markedly reduce the activity of the factor. It could be stored at ?20°C or lyophilized. The synergistic factor displayed properties of an enzyme. It enhanced the hydrolysis of p-nitrophenyl esters of fatty acids with an optimum of pH 9.0. The relative hydrolytic activity increased with increase in number of carbon atoms in the fatty-acid chain from 2 to 8 and gradually decreased with the number of carbon atoms from 10 to 18. Copper sulfate markedly and mercuric chloride completely prevented enhancement of the hydrolysis of butyrate. When the synergistic factor was fed to larvae with mercuric chloride, it did not enhance the nuclear polyhedrosis virus.     

The comparative susceptibilities of Pieris brassicae and P. rapae to a granulosis virus from P. brassicae

A comparison was made of the dosage-mortality responses of larvae of Pieris brassicae and P. rapae to infection by P. brassicae granulosis virus (GV). Bioassays with first, second, third, and fourth-instar larvae of both species revealed a marked difference in susceptibility between instars and between species. Median lethal dosages (LD₅₀s) for P. rapae larvae ranged from five capsules for the first instar to 662 capsules for the fourth instar. With P. brassicae, this range extended from 66 capsules to 2.3 × 10⁷ capsules. The time-mortality responses of the two species were similar when fed virus dosages equivalent to an LD₉₀. Median lethal times (LT₅₀s) ranged from 5 days for first-instar larvae to 7–8 days for fourth-instar larvae. A comparison between a long-established laboratory stock of P. brassicae and a stock recently acquired from the field showed no significant difference in their susceptibility to GV. The implications of the pronounced species differences in susceptibility to GV infection are discussed in relation to the potential field control of P. rapae and P. brassicae.     

Changes in growth and virulence of a nuclear polyhedrosis virus from Choristoneura fumiferana after passage in Trichoplusia ni and Galleria mellonella

An isolate of nuclear polyhedrosis virus from Choristoneura fumiferana was fed to neonate larvae of Trichopulsia ni and Galleria mellonella. It caused infection and mortality in both of these species. After passage in the alternate hosts, the isolate became increasingly virulent for these hosts. The passaged virus retained its infectivity for Choristoneura but diseased larvae did not wilt and at death they were found to contain only a few polyhedra indicating the virus had been changed.     

gfp基因标记的重组杆状病毒对棉铃虫幼虫的侵染历程

 用携带杆状病毒极晚期多角体蛋白基因启动子驱动gfp表达的重组病毒rHa FGP感染棉铃虫三龄幼虫 .在感染后不同时间取样 ,分离不同组织 ,制片 ,置于倒置荧光显微镜下观察基因的表达 .结果发现 ,随着感染时间的推移 ,荧光产生的部位出现更替 ,随后荧光强度也发生相应的变化 .从荧光出现的先后初步推断出杆状病毒对昆虫幼虫的侵染路线 :中肠上皮→血淋巴→气管系统→脂肪体 真皮 .在幼虫感染后 12h荧光即出现于中肠细胞中 ,表明此时已有极晚期蛋白表达 .说明利用杆状病毒极晚期基因启动子驱动苏云金芽孢杆菌杀虫晶体蛋白基因 (cry)表达 ,从而提高杆状病毒的杀虫毒力是可行的     

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.     

Bioassay of a purified nuclear polyhedrosis virus against Heliothis armigera

A precise bioassay method, which is not limited by lack of field applicability, as are peroral administration techniques, is described. Purified nuclear polyhedrosis virus (NPV) suspensions were assayed against third and fourth instar Heliothis armigera larvae to provide standards for additive and field testing. Third instar larvae proved to be approximately one hundred times more susceptible to the NPV disease than fourth instar larvae. The minimum time to mortality was 4 days.     

Developmental cytology of the midgut in the flesh-fly, Sarcophaga bullata (Parker)

The cytological comparisons of the midgut in Sarcophaga bullata (Parker) between the second instar, the third instar larvae and the adult are made. The adult midgut differs from that of the larvae in the following ways: (1) the peritrophic membrane is thicker than in the larvae and has become multi-layered; (2) epithelial cells are smaller; (3) branched microvilli are present throughout the entire midgut instead of being present only in the posterior region as in the larval midgut; (4) nuclear pores are less frequent; (5) lysosome-type structures occur less frequently; (6) the basal membrane is thicker; (7) the z-bands in the surrounding muscle fibers are more distinct in adults. The possible function and the significance of these structures related to previous observations in Sarcophaga and other Diptera are discussed.     

A new cytoplasmic polyhedrosis virus from the salt-marsh mosquito, Aedes cantator (Diptera: Culicidae)

The ultrastructure, development, and histopathology of a new cytoplasmic polyhedrosis virus of Aedes cantator are described. Virus particles measure 70 nm in diameter, are icosahedral in shape, and consist of a central electron-dense core surrounded by a capsid with six projections. Occlusion bodies are irregular in size (0.5–3.0 μm) and shape and contain several virus particles. Virus particles are assembled within an interconnecting network of fine filaments and are occluded by the deposition of a proteinaceous crystal around groups of mature virus particles within a virogenic stroma. Infections are confined to cells of the cardia, gastric ceca, and posterior portion of the midgut, which hypertrophy and frequently lyse. Infected larvae die during the fourth larval instar or as pupae. The prevalence of infection in natural field populations is less than 1%.     

Electron microscope study on the replication of Autographa nuclear polyhedrosis virus and Spodoptera nuclear polyhedrosis virus in Spodoptera exigua

A comparative study of Spodoptera nuclear polyhedrosis virus (NPV) and Autographa NPV replication in Spodoptera exigua revealed some cytopathologic differences. Infection with Spodoptera NPV was accompanied by electron-dense intranuclear granules. Autographa infection within the midgut led to secretion within the lumens of the goblet cells of paracrystalline arrays of small, round particles, 9.5 nm in diameter. Autographa virus was also observed in various stages of possible replication within the cytoplasm.     

A synchronous peroral technique for the bioassay of insect viruses

A relatively fast and simple peroral technique for the bioassay of insect viruses is described in which newly hatched larvae ingest a uniform volume of virus suspension. Three isolates of the Autographa californica nuclear polyhedrosis virus (NPV) and one isolate of the Heliothis zea NPV were used to test the procedure with Trichoplusia ni and H. zea larvae, respectively. Within-assay and between-assay variation was very low with coefficients of variation averaging 0.012 ± 0.006 and 0.20 ± 0.04 for time-mortality and dose-mortality tests, respectively. The synchronous uptake of virus removed the acquisition-time component of the LT₅₀ values while the constant volume improved the accuracy of LD₅₀ values. The procedure was shown to be suitable for a wide variety of lepidopterous species, including Spodoptera frugiperda, S. eridania, Estigmene acrea, Plutella xylostella, Choristoneura fumiferana, Ostrinia nubilalis, Plodia interpunctella, and Pieris rapae.     

Infection of a nucleopolyhedrovirus to Neodiprion zhejiangenis Zhou & Xiao (Hymenoptera: Diprionidae)

Neodiprion zhejiangenis Zhou &; Xiao 1981 (Hymenoptera: Diprionidae) nuclear polyhedrosis virus (NezhNPV) is a biocontrol factor with high development potential and application prospects. To study the insecticidal activity of NezhNPV against the larvae of N. zhejiangenis, the susceptibility of larvae at different instars to different NezhNPV concentrations was determined based on laboratory bioactivity. At 2.0?×?10⁷ polyhedron inclusion body (PIB)/mL, the median lethal time required to kill 50% of the instar 2 and 3 larvae was approximately 5 d, whereas the median lethal time of the instar 4 and 5 larvae was 8–9 d. Interestingly, at the NezhNPV concentrations of 2.0?×?10³ and 2.0?×?10⁴ PIB/mL, the feeding ability of the surviving larvae was decreased by 57.4% and 76.4%, respectively, compared with the controls; the pupal weight at both concentrations was decreased by approximately 27%, and the adult emergence rate was decreased by 27.4% and 50.9%, respectively, with a significantly higher proportion of males than of females. The results showed that younger instar larvae of N. zhejiangenis were more susceptible to NezhNPV infection than older instar larvae. The larval mortality rate was dependent on the larval instar and NezhNPV concentration.