大菜粉蝶颗粒体病毒对菜粉蝶幼虫取食、生长、发育的影响

菜粉蝶幼虫在一、二、三、四龄初期饲以大菜粉蝶颗粒体病毒(PbGV),食量减少99.3—38.4%;四龄末或五龄初饲毒,食量反而比健虫高出36.3%和87.2%。幼虫感病后取食期的缩短或延长是食量变化的主要原因。幼虫病死前脱皮过程先被抑制,其结果往往使得死亡时所处的龄期明显延长。幼虫在四龄末饲毒后,由于五龄期延长,食量增加,其死亡前达到的最高体重平均为357.9毫克,远高于健虫化蛹前的最高体重。PbGV对菜粉蝶化蛹也有明显影响。幼虫在五龄前饲毒,一般不能化蛹。五龄第1—3天饲毒,化蛹率26.4—87.9%,第4天饲毒对化蛹无影响。本文结果对应用PbGv防治菜粉蝶幼虫危害以及对该病毒的大量增殖均具指导意义。     

Localization of juvenile hormone esterase during development in normal and in recombinant baculovirus-infected larvae of the moth Trichoplusia ni.

The pathogenesis and cellular localization of juvenile hormone esterase (JHE) was examined in larvae of the moth Trichoplusia ni, infected with a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus: AcNPV) engineered to produce high levels of JHE (JHE virus). The course of JHE localization in the recombinant virus infected larvae was compared with that of both wild type AcNPV infected, and uninfected larvae, using immunogold electron microscopy. In the JHE virus infected insects, high levels of JHE were observed in the endoplasmic reticulum of all cells showing evidence of viral structures in the nucleus, except for gut cells which showed only background JHE levels. Tracheole cells and haemocytes appeared to play a role in the dissemination of infection. In uninfected larvae, fat body and epidermis were the major tissues staining for JHE, which was only detectable at peak times of JHE activity during the fifth instar: lower levels at other times could not be distinguished from background. JHE was also present in lysosomes of granular haemocytes: these lysosomes increased in number in the fifth instar compared to the fourth instar. Similar lysosome-like granules in the pericardial cells did not become highly positive for JHE antigen until the fifth instar.     

Influence of larval stage and virus inoculum on virus yield in insect host Neodiprion abietis (Hymenoptera: Diprionidae)

Virus yield produced by dead larvae of balsam fir sawfly, Neodiprion abietis (Harris) (Hymenoptera: Diprionidae), that had been infected at four different larval stages (second, third, fourth, or fifth instar) with two virus concentrations (10(5) polyhedral inclusion bodies (PIB) /ml or 10(7) PIB/ml), were analyzed and compared to determine the effects of instar and amount of virus inoculum on virus production. The results indicate that both larval stage and inoculation dosage significantly affect virus yield. On average, each dead larva produced 1.36-12.21 x 10(7) PIB, depending upon larval age and virus concentration of inoculation. Although each dead larva produced more PIB when it was inoculated in the fourth or fifth stage, inoculation of these larvae did not result in the highest virus yield because of low larval mortality. In terms of net virus return, third instars would maximize virus yield when they are inoculated with a virus concentration that can cause 95-100% larval mortality.     

颗粒体病毒在小菜蛾种群中的垂直传播方式的初步观察

在室内观察了小菜蛾颗粒体病毒PlutellaxylostellaGranulosisVirus(RxGV)在小菜蛾种群水平上可能的垂直传播途径。结果表明 :(1 )上代小菜蛾Plutellaxylostella(L .)幼虫期饲毒不能将小菜蛾颗粒体病垂直传递到下代 ;(2 )取食病毒的雌蛾产的卵所孵化的幼虫中 ,没病毒感染出现 ,说明病毒不能经小菜蛾成虫体内传播到下一代。 (3 )用小菜蛾颗粒体病毒处理卵卡时 ,其初孵幼虫有少量被病毒感染死亡 ,浓度高时感染率也较高 ,但感染率均在 1 0 %以下 ,说明小菜蛾颗粒体病毒能经卵表垂直传递给下代 ,但传递效率不高。但当用病毒处理叶片上的卵时 ,初孵幼虫死亡率高达 77 73 % ,而且孵化出的幼虫大多数染病且死于 3龄以前 ,因此田间病毒的施用时间可提早到卵高峰期。     

A Tipula paludosa population with a high incidence of two pathogens

The incidence of lethal parasites in the larvae of a Tipula paludosa population was monitored for two seasons. The proportions of larvae infected with Tipula iridescent virus (TIV) and a tachinid insect were similar to those in previously studied populations, whereas the proportions of larvae infected with Tipula nuclear polyhedrosis virus (NPV) and a spore-forming bacterium (SFB) were higher. Conservative estimates of mortality due to these four agents were 10.7% in 1977–1978 and 7.7% in 1978–1979. The mean population density and the proportion of SFB-infected larvae were lower in 1978–1979 than in 1977–1978, while the proportion of NPV-infected larvae was higher. In 1979 the proportion of NPV-infected larvae was positively correlated with population density, which was highest in the wettest part of the study area. In both seasons the proportion of SFB-infected larvae was negatively correlated with population density. Larvae infected with the NPV or the SFB became pallid at an advanced stage of infection, but, although infected larvae were found throughout the larval period, pallid larvae were only found in the later part. It is suggested that larvae become infected in an early instar, then the infections slowly develop throughout the remainder of the larval period. Five larvae were found with mixed infections; four were infected with the SFB and NPV, while the fifth was infected with the SFB and TIV.     

Biological characterization of an English granulovirus from the summer fruit tortrix moth, Adoxophyes orana

Adoxophyes orana granulovirus (AdorGV) was isolated from overwintering larvae in an orchard in Kent, in the UK. The developmental time of each A. orana instar was determined by measuring the size of the head capsule. The susceptibility of the larvae to the English isolate of AdorGV was evaluated in laboratory bioassays using inoculation by microdroplet feeding and applied dose assays. A series of bioassays were performed to determine LD(50) and ST(50) values for first, fourth and fifth instar larvae. The median lethal doses ranged from 30 occlusion bodies in first instar to 1.36 x 10(6) in fifth instar. The median survival time decreased the later the larvae were infected and ranged from 37 days in first instar to 24 days in fifth instar. Approximately half of the infected larvae released a discharge rich in occlusion bodies from their posterior end prior to death. Approximately 85% of larvae attempted pupation and died as larva-pupa intermediates.     

Characterization of Adoxophyes honmai single-nucleocapsid nucleopolyhedrovirus: morphology,structure, and effects on larvae

A nucleopolyhedrovirus (NPV) was isolated from a diseased larva of the smaller tea tortrix, Adoxophyes honmai, collected from a tea field in Tsukuba, Ibaraki, Japan. Electron microscopic observations confirmed that A. honmai NPV (AdhoNPV) was a single-nucleocapsid type virus. The genome size of AdhoNPV was estimated to be 111.6 +/- 0.9kb (mean +/- SE) by restriction endonuclease analysis. AdhoNPV was also infectious to two other Adoxophyes species, the summer fruit tortrix Adoxophyes orana and Adoxophyes dubia. The LD50 values for neonatal, second, third, fourth, and fifth (final) instar larvae of A. honmai were determined as 61, 107, 688, 1,961, and 4,085 occlusion bodies/insect, respectively. Most of the infected larvae died 5-9 days after molting to the final instar, regardless of the timing of inoculation. However, when neonates were exposed to extremely high doses of AdhoNPV (greater than 100 x LD90), larval development was prevented and most of the larvae died in the first instar.     

The mode of transmission of Tipula iridescent virus: II. Route of infection

Tipula iridescent virus (TIV) was inoculated into Tipula oleracea larvae via different routes. It was found to be much more infective when it was injected into the hemocoel than when it was ingested by the larvae.T. oleracea embryos did not become infected when eggs were laid in agar containing TIV, and there was no evidence that larvae can become infected via the spiracles or that transovum transmission occurs. It is suggested that TIV is transmitted principally by cannibalism, including killing and ingesting infected larvae, and finding dead infected larvae and ingesting them. It is proposed that a new generation becomes infected by first-instar larvae feeding upon infected fourth-instar larvae which have survived from the previous generation.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

Effect of tinopal LPW on the insecticidal properties and genetic stability of the nucleopolyhedrovirus of Spodoptera exigua (Lepidoptera: Noctuidae)

The influence of an optical brightener, Tinopal LPW, on the activity of a purified genotype of the nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner), was determined in second to fifth instar (L2-L5) S. exigua. When mixed with viral occlusion bodies (OB) 1% Tinopal LPW significantly reduced the median lethal dose (LD50) of the virus in all instars compared with insects treated with SeMNPV alone. Levels of enhancement, as determined by LD50 values, ranged from 2.6- to 580-fold, depending on the instar. The greatest enhancement occurred on the two later instars, L4 (70-fold) and L5 (580-fold), which show a much higher resistance to SeMNPV infection than earlier instars. The median time to death (MTD) values were not significantly different in any instar among larvae treated with SeMNPV + Tinopal LPW and those treated with SeMNPV alone. Larval development in SeMNPV + Tinopal LPW treated larvae was retarded, in second and fourth instars, compared with controls or larvae treated with SeMNPV alone. The OB yields from SeMNPV treated larvae were almost 1.6-fold greater in second instars (9.3 x 10(6) OBs/larvae), and 1.9-fold greater in fourth instars (1.9 x 10(8) OBs/larvae), than those obtained in larvae treated with SeMNPV + Tinopal LPW. The addition of 1% Tinopal LPW to the virus suspension did not alter the genotypic composition of viral progeny during four successive passages of the virus.     

A Biochemical Evidence of the Inhibitory Effect of Diflubenzuron on the Metamorphosis of the Silkworm,Bombyx mori

Diflubenzuron (DFB) has been known to prevent metamorphosis of silkworm, Bombyx mori, from larval to pupal stage at low dose exposure. To explain this inhibitory action of DFB, a hypothesis was raised that DFB acts like juvenile hormone (JH) or DFB inhibits JH esterase to increase endogenous JH titer. A JH bioassay using isolated abdomen clearly indicates that DFB does not act as JH analog because DFB did not induce vitellogenesis in the isolated female abdomen, while endogenous JHs did significantly. General esterase activities in hemolymph were lower in DFB-treated fifth instar larvae than in the control larvae, but there was no difference between fat body esterase activities in both groups. Two hemolymph esterases (‘E1’ and ‘E2’) of the fifth instar larvae were separated and visualized by α-and β-naphthyl acetate. From in vitro incubation experiment, the cathodal esterase (‘E1’) was sensitive to DFB at its nanomolar range. Considering the fact that early fifth instar larvae have high level of JH esterase in the hemolymph, these results suggest that DFB inhibit larval to pupal metamorphosis by blocking JH degradation, which increases endogenous JH titer especially at the critical period when the larvae determine metamorphic development at the following molt.     

The mode of transmission of Tipula iridescent virus: I. Source of infection

A virus was isolated from a diseased tipulid larva and identified as Tipula iridescent virus (TIV) on the basis of the size and morphology of the virion, the production of iridescence in vitro and in infected tipulid larvae, and a serological reaction between antiserum against the virus and an isolate of TIV.A stock of Tipula oleracea was bred in the laboratory. Subjection of larvae to several stress factors did not result in any evidence for activation of a latent virus. Healthy T. oleracea larvae did not develop iridescence when confined in petri dishes with either live TIV-infected larvae or with large amounts of their feces, although these feces were found to contain infective virus by injecting extracts into healthy larvae. It appears that the concentration of virus in the feces of infected larvae is not high enough for them to serve as a source of infection. It was shown that the cadavers of TIV-infected larvae can serve as a source of infection for healthy first- and fourth-instar larvae.     

Nanoparticle-induced morphological transition of Bombyx mori nucleopolyhedrovirus: a novel method to treat silkworm grasserie disease

Grasserie, a polyorganotrophic disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV), accounts for lethal infection to fifth instar silkworm larvae. It was found that nanoparticle (NP)-induced morphological transformation of BmNPV polyhedra could reduce the infectivity of BmNPV both in cell line and in silkworm larvae. Initially, 11 NPs were screened for evaluation of their nature of interaction with polyhedra surface through scanning electron microscopy. Amongst these NPs, lipophilically coated silica nanoparticle (SNPL), alumina nanoparticles in the hexagonal close-packed α structure and aspartate capped gold nanoparticle transformed polyhedra were tested for their infectivity in B. mori cell line using cytopathic effect and plaque reduction assay. SNPL was evaluated for its bio-efficacy in fifth instar silkworm larvae. The study of polyhedra morphology as a function of NP concentration showed severe ‘roughening’ of the polyhedra with replacement of the regular facets by a large number of irregular ones by SNPL, and this caused transition of highly infectious polyhedra into a nearly spherical, non-infectious structure. A moderate polyhedra roughening was observed for alumina NPs, and no roughening was noticed for gold NPs. The morphological changes could be correlated with reduction of virus-induced cytopathic effect and plaque formation, and increased survival rate of SNPL transformed polyhedra infected silkworm larvae to 70.09?±?6.61 % after 96 h. In this group, 61.04?±?8.03 % larvae formed normal cocoons from which moths eclosed, laid eggs and larvae emerged. This study could lead to open up newer pathways for designing nano pharmaceuticals to combat other viral diseases.     

Infectivity, speed of kill, and productivity of a baculovirus expressing the itch mite toxin txp-1 in second and fourth instar larvae of Trichoplusia ni

A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and inserted into the genome of the Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombinant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV-C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD(50) values between the recombinant virus and its wild-type parent strain but, as expected, the LD(50) was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50-60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approximately 60 h and dying 10-20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompanied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied between the different viruses, concentrations, and instars. The yield per unit weight of the larvae was found to be constant at a low virus dose and increased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations.     

Changes in Trace Metals in Hemolymph of Baculovirus-Infected Noctuid Larvae

We studied how biologically relevant trace metals (i.e., micronutrients) in the hemolymph of larval Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae) changed in response to per os baculovirus infection, larval development, and injection of heat-killed bacteria. Concentrations of hemolymph Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, and Zn were measured using inductively coupled plasma-mass spectrometry. H. virescens larvae exhibited greater fluctuations in hemolymph trace metal levels in response to baculovirus infection and development than did H. zea larvae. H. zea single nucleopolyhedrosis virus infection significantly altered the levels of Cu, Fe, Mg, Mn, Mo, and Zn in fourth instar H. virescens larvae. Conversely, in fifth instar H. virescens and both H. zea instar infections, no metal levels were significantly different between infected and uninfected larvae. In fourth instar H. virescens hemolymph, Cu, Fe, Mo, and Zn increased during development. Cu, Fe, Mg, Mn, Mo, and Zn levels changed significantly during development in fifth instar H. virescens as well as both H. zea instars. Based on this analysis, metals were identified whose levels changed during development in both species and during the immune response of H. virescens larvae.     

Effects of diet on development of Podisus nigrispinus (Dallas) (Het., Pentatomidae), a predator of the cotton leafworm

Abstract:  The effects of diet on development of Podisus nigrispinus (Dallas) (Het., Pentatomidae) were studied at 25°C, relative humidity of 60 ± 10%, and photoperiod of 14 h light : 10 h dark. Development times of P. nigrispinus nymphs were similar when fed with third or fifth instar larvae of cotton leafworm ( Alabama argillacea Hübner) (Lep., Noctuidae) or Tenebrio molitor L. (Col., Tenebrionidae). When fed with housefly larvae ( Musca domestica L.) (Dipt., Muscidae) or artificial diet, the predator had a longer development time. Independent of diet, instar or sex, the females of P. nigrispinus showed a longer longevity than the males. The total survival of the nymphal stage, on the different diets, varied from 22.46 (fed with housefly) to 77.33% (fed with T. molitor larvae). P. nigrispinus males were heavier when fed with third or fifth instar cotton leafworm larvae than when fed with artificial diet. The weight of the females varied from 37.91 (with artificial diet) to 64.68 mg (with fifth instar cotton leafworm larvae). Independently of the diet, newly emerged females of P. nigrispinus were heavier than the males. Females of P. nigrispinus which were fed with fifth instar cotton leafworm larvae had heavier ovaries than those fed other diets.     

Infection by the microsporidium Vairimorpha necatrix (Microspora: Microsporidia) elevates juvenile hormone titres in larvae of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae)

The effects of infection by a microsporidium, Vairimorpha necatrix (Kramer), on the endogenous levels of juvenile hormones in tomato moth (Lacanobia oleracea L.) larvae were investigated. Levels of juvenile hormone II (JH II) were 10-fold greater in the infected larvae on day two of the sixth stadium but no significant difference was observed on day seven. Juvenile hormone I (JH I) was also detected in day two and day seven sixth instar infected larvae but was not detected in non-infected larvae. The duration of the fifth and sixth stadia was significantly longer for infected larvae when compared with non-infected larvae. No evidence was found to suggest that supernumerary moults are a feature of infection by V. necatrix in L. oleracea larvae. Experiments were performed to determine whether the elevation in JH levels, which probably prevents pupation, is an adaptive mechanism of the microsporidium for extending the growth phase of the host, thereby allowing increased spore production. A proportion of infected larvae were collected on days 9 and 24 of the sixth stadium and spore extracts prepared from each larva. These days represent the average duration of the sixth stadium required for uninfected larvae to reach pupation, and the average number of days that V. necatrix-infected larvae survive in the sixth stadium before dying from infection. The mean spore yields from infected larvae 24 days into the sixth stadium were significantly higher than the spore yields obtained from day nine sixth instar larvae. The hypothesis that V. necatrix manipulates host endocrinology (i.e. prolong the host larval state to maximise spore yield) is discussed in context with the results obtained.     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

Interaction between the tachinid parasite,Sturmiopsis inferens and granulosis virus in the sugarcane shoot borer,Chilo infuscatellus [Lep.: Crambidae]

Competition between granulosis virus (GV) and the larval parasite,Sturmiopsis inferens Tns. (Tachinidae: Diptera), was studied in 3^rd — and 4^th — instar larvae of the sugarcane shoot borer,Chilo infuscatellus Snellen (Crambidae: Lepidoptera), under laboratory conditions. Mortality due to GV infection and parasitization was 76.8 and 47.6 per cent, respectively, when they were tested separately. But when hosts were infected simultaneously with microfeeding of GV and larval parasite, a significantly low parasitism (5.5%) was obtained compared to 74.8 per cent mortality by GV infection. When the larvae were microfed with the GV 6 days after inoculation with parasitic maggots, mortality due to the virus was reduced significantly to 20.5 per cent, but when the maggot inoculation was preceded by virus microfeeding 6 days before, parasitization was unsuccessful, while 75% of larvae died of virus. Results obtained from field — collected larvae also showed that significantly more parasite puparia were recovered from healthy larvae than from virus — infected larvae. Similar differences in parasitization were not obtained in the case of healthy or virus — infected pupae.