Genetic basis for resistance to a granulosis virus in the potato moth Phthorimaea operculella

Evidence is presented to show that the resistance to a granulosis virus in a laboratory strain of potato moth, Phthorimaea operculella, is controlled by a single dominant autosomal gene which segregates according to simple Mendelian ratios. This may be only one of a number of genes which influence the phenotypic expression of resistance and contribute to the wide variability in response to the virus shown by field populations of the moth.     

Inhibition of cuticle production in imaginal discs of Plodia interpunctella (cultured in vitro): Effects of colcemid and vinblastine

The relationship of the microtubular system to the production of cuticle was evaluated by culturing wing imaginal discs from Plodia interpunctella in medium that contained 20-hydroxyecdysone and either colcemid or vinblastine. Examination of the treated tissue with both a dissection stereomicroscope and an electron microscope showed that the microtubule inhibitors prevented the formation of cuticle. The inhibitors also prevented the synthesis of chitin, but did not reduce protein synthesis. These results support the hypothesis that the secretion of cuticular components by insect cells requires the integrity of the microtubular system.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

Environmental triggers to cryoprotectant modulation in separate populations of the gall fly, Eurost a solidaginis (Fitch)

Northern and southern populations of the gall fly Eurosta solidaginis utilize quantitatively distinct adaptive strategies when exposed to a laboratory simulation of winter temperatures. Both populations are freezing tolerant and rely in part on the temperature-dependent accumulation of glycerol and sorbitol, and static but elevated levels of trehalose for protection. The accumulation triggers are time-temperature dependent. For northern and southern populations, exposure to 5°C for periods exceeding 24 hr, but less than 5 days or 5°C with a gradual reduction (1°C/day) results in the accumulation of sorbitol at 1.5 μg/mg/day. Glycerol levels remain essentially constant between 10° and ?25°C in each population. However, the concentration of glycerol in the Minnesota population is 3–4 times greater that that of the Texas specimens. Haemolymph melting points varied quantitatively with changing cryoprotectant levels. No significant difference was noted between the supercooling points of each population. This suggests that ice-nucleator levels were comparable throughout the exposure period.     

Protein components of two strains of granulosis virus of the armyworm, Pseudaletia unipuncta (Lepidoptera,Noctuidae)

A synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain of a granulosis virus (GV) infect the armyworm, Pseudaletia unipuncta. The protein components of the enveloped virions and of the capsule (inclusion body) were compared between the two strains. When the enveloped virions of both strains were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the protein patterns were similar except for minor peaks in the higher molecular weight region. On the other hand, the capsule proteins differed between the two strains when examined with immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. The capsule proteins of GVH were composed of two major proteins, one a structural protein and the other the protein of the synergistic factor. The capsule protein of GVO, however, had only the structural protein. The rocket immunoelectrophoresis and affinity chromatography indicated that the structural proteins of the two strains were partially dissimilar. The molecular weight of the structural protein of GVO was 29,100 ± 500 and that of GVH was 28,700 ± 500. The amount of synergistic factor in a GVH capsule was about 5% of the dissolved capsule components.     

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.     

Effect of a nuclear polyhedrosis virus on the relationship between Trichoplusia ni (Lepidoptera: Noctuidae) and the parasite, Hyposoter exiguae (hymenoptera: Ichneumonidae)

The effect of a nuclear polyhedrosis virus on the relationship between Trichoplusia ni and the parasite, Hyposoter exiguae, was investigated to determine if the virus could invade and multiply in the tissues of the parasites, if parasites which emerged from virus-infected T. ni larvae had normal emergence, fecundity, and longevity, and if the parasite could serve as a vector for the virus. Light microscopy revealed particles which appeared to be polyhedra within the lumen of the midgut of parasite larvae from virus-infected hosts. Transmission electron microscopy confirmed the presence of polyhedra and free virions within the midgut of the larvae. Polyhedra or free virions were never found within any parasite tissues. Parasite larvae within hosts exposed to virus before parasitization perished when their hosts died of virus infection. Parasite larvae in hosts exposed to virus after parasitization completed their development before their hosts died of virus infection. The proportion of parasites which survived increased as the time between host parasitization and host virus exposure increased. Parasite larvae which developed in hosts exposed to the virus soon after parasitization spent significantly less time in their hosts than did parasites which developed in noninfected hosts. There was no significant difference in time spent in the pupal stage, percent adult emergence, adult longevity with and without food and water, and fecundity of parasites which developed in virus-infected hosts and those which developed in noninfected hosts. Female parasites laid as many eggs in virus-infected hosts as they did in noninfected hosts. Sixty percent of the female parasites which oviposited in virus-infected hosts vectored infective doses of virus to an average of 6% of the healthy hosts subsequently exposed to them. None of the healthy host larvae exposed to male parasites which had been exposed to virus-infected host larvae became infected with the virus. Forty percent of the female parasites which developed in virus-infected hosts transmitted infective doses of the virus to an average of 65% of the healthy host larvae exposed to them. Ninety percent of the male parasites which developed in virus-infected hosts transferred infective doses of the virus to an average of 21% of the healthy host larvae exposed to them.     

Factors controlling in vitro hatching of Vairimorpha plodiae (Microspora) spores and their infectivity to Plodia interpunctella,Heliothis virescens, and Pieris brassicae

Factors which influence the hatching of spores and proliferation of stages of the microsporidium Vairimorpha plodiae in two susceptible insects, Plodia interpunctella and Heliothis virescens, and one nonsusceptible insect, Pieris brassicae, were investigated. Spores hatched in 0.1 and 1 m KCl solutions when subjected to a change in pH, from pH 11 to pH 8. K⁺ was essential for hatching; NaCl solutions were not effective. Ca²⁺ and Mg²⁺ inhibited hatching, and calcium and magnesium chelating agents enhanced it. All three insect species had alkaline midgut contents and smooth, fragile peritrophic membranes. Spores hatched inside the midguts of all three insect species (P. interpunctella: maximum rate, 92.5%; H. virescens, 91.5%; P. brassicae, 82%). Sporoplasms were observed in the midgut epithelial and associated tracheole cells of P. brassicae. Both H. virescens and P. brassicae became infected when injected intrahemocoelically with spores.     

Site of action of a synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor (SF), which is present in the capsule matrix protein of a granulosis virus of the armyworm, Pseudaletia unipuncta, enhances baculovirus infection in armyworm larvae. The site of action of the SF was investigated. The oral inoculation of SF did not enhance the infectious hemolymph virions which had been inoculated into the hemocoel. The SF also did not enhance the infection of purified enveloped virions when both virus and SF were inoculated into the hemocoel, but enhancement occurred when they were inoculated orally. Thus, the activity of the SF was confined to the midgut lumen. Observations with ferritin-conjugated antibody indicated that the site of action of SF was the cell membrane of the microvillus. There were more ferritin particles attached to midgut cell membranes of larvae inoculated orally with SF than to those of control larvae inoculated with buffer.     

The susceptibility of the pea moth, Cydia nigricana, to infection by the granulosis virus of the codling moth, Cydia pomonella

Laboratory studies demonstrated that neonate larvae of the pea moth, Cydia nigricana, are susceptible to infection with a granulosis virus (CpGV) isolated from the codling moth, Cydia pomonella. Comparative LC₅₀ values for C. nigricana and C. pomonella are 1.90 × 10⁵ and 1.54 × 10⁴ capsules/ml of diet, respectively. The virus extracted from CpGV-infected pea moth larvae is serologically related, and probably identical, to CpGV.     

Plasmatocyte sensitivity to plasmatocyte spreading peptide (PSP) fluctuates with the larval molting cycle

Plasmatocyte spreading peptide (PSP) is a cytokine from the moth Pseudoplusia includens that activates a class of hemocytes called plasmatocytes to bind and spread on foreign surfaces. Previous structure-function studies on PSP used plasmatocytes collected from P. includens larvae that were in the late stages of the last (fifth) instar. Here, we report that plasmatocyte sensitivity to PSP varied significantly during the fourth and fifth instar. PSP weakly activated plasmatocytes early in the instar when hemolymph juvenile hormone (JH) titers were relatively high and ecdysteroid titers were low, but strongly activated plasmatocytes late in the instar after JH titers declined and ecdysteroid titers rose. In contrast, plasmatocytes did not vary in their response to plasma, which contains other factors besides PSP that affect plasmatocyte function. In vitro assays indicated that 20-hydroxyecdysone (20E) dose-dependently synergized PSP activity, whereas the JH analog methoprene antagonized PSP activity. Methoprene had no effect on adhesion and spreading of granular cells, but plasmatocytes from larvae topically treated with methoprene exhibited a reduction in sensitivity to PSP. Collectively, these results indicate that plasmatocyte sensitivity to PSP fluctuates in relation to the molting cycle, and that PSP activity is affected by juvenoids and ecdysone.     

The transmission of a granulosis virus following the contamination of Pieris brassicae adults

The granulosis virus of Pieris brassicae, when applied as a paste to the tip of the abdomen of females, was readily transmitted to their progeny. Mortality of the progeny decreased with increasing time after treatment from 75.6% immediately after contamination to 2.0% on day 16. The overall mortality of progeny was 31.6%. Transmission following contamination of the tip of the male abdomen with a virus paste was less, with a peak of 11% mortality of progeny on day 2. The maximum mortality of progeny following contamination of the female abdomen with a virus dust was 19.3% with an overall mortality of 9.5%. There was no transmission following the contamination of the male abdomen with a virus dust.     

Attalea phalerata and Bactris glaucescens (Arecaceae, Arecoideae): Phenology and pollination ecology in the Pantanal, Brazil

We compared the reproductive and vegetative phenology and pollination ecology of sympatric Attalea phalerata and Bactris glaucescens palms (Arecaceae) in the Pantanal, Brazil, in a riparian forest area subject to periodic flooding. Attalea phalerata has a solitary stem and produces staminate, pistillate and rarely bisexuals inflorescences that open during the day. Bactris glaucescens has multiple stems and has bisexual flowers with crepuscular/nocturnal anthesis. Both species present bud break and leaf-budding during the year. Attalea phalerata shows continual annual flowering with ripening of fruits during the dry season. For B. glaucescens flowering occurs simultaneously with fruiting for about seven months, and fruit production can be influenced by temperature and level of flooding. The difference in the timing of anthesis of the flowers ensures production of floral resources during both day and night when both species of palms are flowering. The floral structures of both species have morphological characteristics associated with pollination by insects (mainly beetles), such as the presence of odor, stigmatic secretion, heat production, and adhesive substances (“pollenkitt”) in the pollen. In A. phalerata, the main pollinators were Mystrops sp. (Nitidulidae) and Madarini (Curculionidae). Derelomus sp. (Curculionidae) and Paratenetus sp. (Tenebrionidae) beetles visited B. glaucescens during the day and could have pollinated the flowers at these occasions.     

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean ( Vigna radiata L. Wilczek)

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC₂₀ generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC₂₀F₂ population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2 cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC₂₀F₂ population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2 cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene. Received: 20 November 1997 / Accepted: 6 January 1998     

Descriptive models for the system of the natural spread of infection of rice dwarf virus (RDV) by the green rice leafhopper,Nephotettix cincticeps Uhler (Hemiptera: Deltocephalidae)

Seasonal changes in the densities of eggs and of 1st instar nymphs of N. cincticeps, a vector of the rice dwarf virus (RDV) and those in the proportion of virus-infected rice hills were examined by regular field censuses during the period of 1964–69 at two localities in Kochi, Nangoku and Ino. The proportion of virus-infected insects in the vector population was measured by the serological test. Mean fecundity of adult females of N. cincticeps in different generations was assessed by the method proposed byHokyo andKiritani (1968) in 1966 and 1967. Using observed variables as mentioned above, two mathematical models were built up for the system of the natural spread of infection of RDV. Submodels describing generation-to-generation changes in densities of eggs, 1st instar nymphs and leafhopper adults were involved in both Models 1 and 2. In Model 1, the proportion (P) of infected insects in the vector population was assumed to be determined directly by the amount (I_v) of infected rice plants in the field. In Model 2, instead of relating P directly to I_v,P was related to the acquisition rate (W) of RDV by the insects and to the effect of RDV (αr) on fecundity and survival of infected insects. Then, W was related to I_v. The estimates of P by these models fitted well to the observed values for 1st to 3rd generations but a great deviation was noted in 4th and 5th generations. For the percentage of infected rice hills, both models gave relatively good estimates in the early planted rice, though they gave overestimated values in the late planted rice.