Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Effect of temperature and humidity on infection by Beauveria bassiana,Metarhizium anisopliae, and Paecilomyces farinosus

Larvae of the elm bark beetle, Scolytus scolytus, were inoculated with conidia of the entomogenous fungi Beauveria bassiana (two strains), Metarhizium anisopliae (two strains), and Paecilomyces farinosus (two strains) and incubated over a range of temperatures (2°, 6°, 10°, 15°, and 20°C). One strain each of B. bassiana and P. farinosus caused infection even at 2°C, whereas the two strains of M. anisopliae caused no infection below 10°C. Infection of adult beetles by B. bassiana (one strain) and M. anisopliae (one strain) was tested at 15°, 20°, and 25°C (B. bassiana) and at 15° and 20°C (M. anisopliae). Fungal infection occurred at all three temperatures, but at 25°C beetles tended to succumb to bacterial infection. The effect of relative humidity on infection of larvae by B. bassiana (one strain), M. anisopliae (one strain), and P. farinosus (one strain) was tested at 51, 74, 86, 90, 95, 97.5, and 100% relative humidity. B. bassiana and M. anisopliae caused some infection at all humidities: with P. farinosus there was no infection at the two lowest humidities. Mortality due to infection by these fungi was most rapid at the highest humidities.     

Reversible envelope effects during and after killing of Escherichia coli W by a highly-purified rabbit polymorphonuclear leukocyte fraction

The effects of a highly-purified, potently bactericidal fraction from rabbit polymorphonuclear leukocytes on the envelope of Escherichia coli (W) have been examined. This leukocyte fraction has equally enriched bactericidal, permeability-increasing and phospholipase A₂ activities, and is essentially devoid of lysozyme, myeloperoxidase and protease activities (Weiss, J., Franson, R.C., Beckerdite, S., Schmeidler, K. and Elsbach, P. (1975) J. Clin. Invest. 55, 33–42). Rapid killing of E. coli by this fraction is accompanied by two almost immediate alterations in the bacterial envelope: (1) a discrete increase in envelope permeability (measured by inhibition of bacterial leucine incorporation by normally impermeant actinomycin D), and, (2) hydrolysis of ¹⁴C-labeled fatty acid-prelabeled E. coli phospholipids. Both envelope effects are promptly reversed during further incubation at 37 °C, but not at 0 °C, with 40 mM Mg²⁺. Reversal is also produced by Ca²⁺ (40 mM) and trypsin (200 μg/ml), but 200 mM K⁺ causes only partial recovery and Na⁺ and hyperosmolar sucrose are ineffective. Upon addition of Mg²⁺, phospholipid degradation ceases abruptly and the labeled products of hydrolysis (free fatty acids and lysocompounds) disappear with a corresponding reaccumulation of radioactive diacylphosphatides. The time course of resynthesis of phospholipids coincides with that of restoration of the permeability barrier. Higher concentrations of the leukocyte fraction and prolonged incubation increase both the extent of phospholipid degradation and the time required for reversal of both envelope effects. These findings suggest that both the initiation of the increased permeability and its reversal are linked to respectively the breakdown and resynthesis of major E. coli membrane phospholipids, and thus depend on the fact that the biochemical apparatus of E. coli remains capable of biosynthesis despite loss of viability.Treatment of E. coli, exposed to the leukocyte fraction, with albumin results in extracellular sequestration of the products of hydrolysis and also restores the permeability barrier to actinomycin D, suggesting that the accumulation of lytic products of lipid hydrolysis within the bacterial envelope, rather than the loss of phospholipids per se, causes increased permeability.     

Pathogenicity of entomopathogenic hyphomycetes, Paecilomyces fumoso-roseus and Nomuraea rileyi, to eggs of noctuids, Mamestra brassicae and Spodoptera littoralis

Bioassays were conducted to determine the susceptibility of egg masses of Mamestra brassicae and Spodoptera littoralis to different spore doses of Paecilomyces fumoso-roseus and Nomuraea rileyi at 20° and 25°C. P. fumoso-roseus was highly virulent against eggs, whereas N. rileyi provoked only a deferred mortality of larvae hatched from treated eggs. Nevertheless, larval mortality of S. littoralis caused by N. rileyi at 25°C was more effective after first-instar larval contamination than after egg mass treatment. The duration of the egg stage could explain differences of susceptibility between the two noctuids at 25°C. Scanning electron microscopical observations suggested two ways of contamination of newly hatched larvae. First, fungal germinations on the chorion surface suggested that newly hatched larvae might be infected by penetration of the egg integument before hatching. Second, conidia on the egg cuticle could be an entomopathogenic inoculum for newly emerging larvae which fed upon chorions. Results showed that pathogenicity of Hyphomycetes to noctuid eggs might be a promising area of investigation for biological control.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Structural Characterization of the Glycoprotein GP2 Core Domain from the CAS Virus, a Novel Arenavirus-Like Species

Fusion of the viral and host cell membranes is a necessary first step for infection by enveloped viruses and is mediated by the envelope glycoprotein. The transmembrane subunits from the structurally defined “class I” glycoproteins adopt an α-helical “trimer-of-hairpins” conformation during the fusion pathway. Here, we present our studies on the envelope glycoprotein transmembrane subunit, GP2, of the CAS virus (CASV). CASV was recently identified from annulated tree boas (Corallus annulatus) with inclusion body disease and is implicated in the disease etiology. We have generated and characterized two protein constructs consisting of the predicted CASV GP2 core domain. The crystal structure of the CASV GP2 post-fusion conformation indicates a trimeric α-helical bundle that is highly similar to those of Ebola virus and Marburg virus GP2 despite CASV genome homology to arenaviruses. Denaturation studies demonstrate that the stability of CASV GP2 is pH dependent with higher stability at lower pH; we propose that this behavior is due to a network of interactions among acidic residues that would destabilize the α-helical bundle under conditions where the side chains are deprotonated. The pH-dependent stability of the post-fusion structure has been observed in Ebola virus and Marburg virus GP2, as well as other viruses that enter via the endosome. Infection experiments with CASV and the related Golden Gate virus support a mechanism of entry that requires endosomal acidification. Our results suggest that, despite being primarily arenavirus like, the transmembrane subunit of CASV is extremely similar to the filoviruses.     

Electron and proton transfers from P-430 to ferredoxin-NADP-reductase in Chlorella cells

After blocking Photosystem II on whole Chlorella cells, we measured the absorption changes between 0°C and ?10°C.The absorption changes measured 2 μs after the beginning of a Xenon Flash are the sum of changes due to P⁺-700 and changes due to P^?-430 (after the subtraction of the carotenoid triplet change and of the electrochromic effect).The reduction of P^?-430 is not resolved by our technique. Its reoxidation presents a half-time around 1 μs at 0°C and around 2 μs at ?10°C.The reduction and protonation of ferredoxin-NADP-reductase to its neutral semi-quinoid form FNRH° present a half-time of about 3 μs at 0°C and 6 μs at ?10°C.The presence of only one photoreducible ferredoxin-NADP-reductase per Photosystem I center is confirmed. The acceptor preceding ferredoxin-NADP-reductase is not ferredoxin, but is an acceptor X' the differential extinction coefficients of which are weak or null from 420 nm to 480 nm.Tentative explanations which would reconcile these results with what was already known about ferredoxin are proposed.     

The in vitro culture of the blood stages of Plasmodium berghei

Plasmodium berghei was cultured in medium 199 with 10% serum at various temperatures. At 31° and 37°C the parasite numbers decreased. Experiments at 15°C achieved consistent multiplication rates greater than one, with a maximum of three-fold.     

Effects of fluctuating temperature and immersion on asexual reproduction in the intertidal sea anemone Hauplanella luciae (Verrill) in laboratory culture

Haliplanella luciae (Verrill) was maintained in laboratory cultures according to a 2 × 2 factorial design. The factors were 1) constant immersion or continuously alternating periods of 6 h immersion followed by 6 h emersion and 2) 18 °C constant temperature or 24 °C during one 6-h emersion period each day and 18 °C for the remaining 18 h. Significantly different numbers of fissions were observed between both the two immersion and two temperature conditions. A negative correlation was found between the number of fissions and the total dry weight of tissue per bowl. There was a different distribution of individual weights in the two immersion conditions. Some instances of pedal laceration were observed.     

Effects of temperature and host age upon the encapsulation of Metaphycus stanleyi and Metaphycus helvolus eggs by brown soft scale Coccus hesperidum

Encapsulation of eggs inserted by Metaphycus stanleyi (Hymenoptera: Encyrtidae) into the brown soft scale Coccus hesperidum (Homoptera: Coccidae) became more frequent as the host matured. This occurred with both laboratory reared and field-collected parasites. After parasitism for 24 hr at 27°C, encapsulation frequency did not differ in hosts reared at 20° or at 27°C, but significantly increased in hosts reared at 33°C. When parasitism and rearing were carried out at the same temperature, the percentage of eggs encapsulated increased from 48.7% at 27°C to 94.1% at 33°C. With M. helvolus, the percentage of eggs encapsulated was considerably higher than with M. stanleyi; e.g., 99.3 vs 48.7%, respectively, at 27°C. At 20° and 27°C, some M. helvolus development occurred in the larvae of brown soft scale but none at 33°C; the adult stages of the host encapsulated all the parasite eggs at these temperatures.     

The growth of four species of Azolla as affected by temperature

The growth of 22 strains of Azolla pinnata R. Br., 3 strains of A. filiculoides Lam. and one strain each of A. mexicana Presl and A. caroliniana Willd. was tested separately in liquid culture media kept in controlled, artificial light (30 klux) growth cabinets. Three temperature levels were used: 33°C (37/29°C day/night), 29°C (33/25°C) and 22°C (26/18°C)/ Photoperiod was 12 h a day.For most A. pinnata strains (except three) and an A. mexicana strain the maximum weekly relative growth rate was higher at 33°C than at 22°C, but not for A. filiculoides and A. caroliniana. The highest value of maximum relative growth rate corresponded to 1.9 doubling days and in most strains this occurred in the first week. As the plants grew, the growth rate slowed down more severely at higher temperatures. The maximum biomass was higher at 22°C than at 33°C in all strains. At 22°C, it took 30–50 days to attain maximum biomass and the highest value was 14 g N m^?2 or 320 g dry m^?2 by A. caroliniana, followed by 12 g N m^?2 or 290 g dry wt. m^?2 by one strain of A. filiculoides. At 29°C, the maximum biomass was attained in 20–35 days. The highest value was 6.3 g N m^?2 or 154 g dry wt. m^?2 by A. caroliniana. At 33°C, most A. pinnata strains gave a maximum biomass of less than 4 g N m^?2 after 13–23 days, while some strains grew up to 30 days, resulting in a higher maximum biomass. The highest maximum biomass at 33°C was 5.5 g N m^?2 or 140 g m^?2 dry wt. by A. pinnata from Cheng Mai while the maximum biomass of A. filiculoides and A. caroliniana was much less. Azolla filiculoides requires lower temperature than other species for its growth. Azolla pinnata has the best tolerance to high temperatures among the four species. Azolla mexicana could not be discriminated from A. pinnata in its response to temperature. Azolla caroliniana may keep an intermediate position between A. filiculoides and A. pinnata in temperature response.The formation of ammonia in the medium was examined and it occurred mostly under stationary growth conditions, but, at 33°C, some strains of A. pinnata and A. mexicana released or formed ammonia at 0.3–0.8 μg N ml^?1 per week during their initial exponential growth stage.     

Transition temperatures in the sensitivity of escherichia coli B/r to lysozyme

Lysozyme attacked Escherichia coli B/r in the absence of EDTA or imposed osmotic shocks when the cells were rapidly cooled below specific temperatures. Cells subjected to lysozyme while being cooled to below 20°C began to lose ability to subsequently form colonies. This sensitivity increased with decreasing temperatures and almost all cells cooled to 0°C were affected. Slightly hypertonic solutions did not improve survival. Cells cooled first to as low as 5°C and then subjected to lysozyme while cool did not lose their ability to form colonies subsequent to rewarming. However, 70% of the cells cooled first to 0°C and subjected to lysozyme lost their colony-forming ability. Cell lysis also began when treated near 5°C, but even when treated at 0°C about 50% of the cells maintained their rod shape in the presence of lysozyme. These results are discussed in terms of a possible phase transition in a portion of the cell envelope and/or a transient osmotic swelling as a results of metabolic pumps failing at the low temperatures.     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.     

A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

Analysis of the survival of Venezuelan equine encephalomyelitis virus and possible viral simulants in liquid suspensions

Aims: To compare the inactivation rate of Venezuelan equine encephalomyelitis (VEE) virus in liquids to that of Sindbis virus (SV, another alphavirus) and to a bacteriophage (MS2) generally used as a viral simulant in the development of countermeasures in biodefense. Methods and Results: Viruses were inoculated into liquids and viral titres were determined at various times postinoculation. The viruses were stable in distilled-deionized (dd) water at 4°C during the 21 days of the study. The inactivation rates of VEE and SV in dd water at 21 and 30°C were very similar (between 0·12 and 0·14 log₁₀ per day), while MS2 was three-fold slower. In tap water (chlorine content between 4 and 5 ppm) at 21°C, VEE and SV were inactivated at twice the rate measured in dd water. Conclusions: The inactivation rates of VEE and SV were similar to each other and faster than MS2 in all liquids tested. Significance and Impact of the Study: VEE is likely to remain viable for many days after release into water, snow, or even chlorinated tap water. SV can be used to estimate the persistence of VEE in liquids, but using MS2 as a simulant would overestimate of the stability of VEE.     

Abortive bacteriophage T4 head assembly in mutants of Escherichia coli

We describe two mutants (tabB-212 and tabB-127) of Escherichia coli K12 in which T-even phage production is temperature-sensitive. Both mutants are linked to purA and may identify a single new bacterial gene tabB. The uninfected bacterium is indistinguishable from wild type at both 30 °C and 42.4 °C. Sodium dodecyl sulphate—polyacrylamide gel electrophoresis of labelled extracts of tabB mutants infected by T4 wild-type phage shows that the modification of viral head precursors (Laemmli, 1970) does not occur, indicating that capsid formation is blocked. The effect is reversible with at least one of the tabB mutants: a shift to 30 °C leads to the cleavage of a significant fraction of precursors synthesized at 42.4 °C.Two classes of T4 mutants are described: one (com^B) which grows on tabB even at 42.4 °C, the other (k^B) which fails to grow on tabB even at the permissive temperature. Both mutants map in T4 gene 31, suggesting an interaction between gene 31 and tabB products.Since gene 31 mutants lead to the random aggregation of head precursors (Laemmli, 1970), we argue that a host product is involved in the ordered polymerization of T4 proteins into capsids or capsid-related structures.     

Functional differences between occluded and nonoccluded viruses of a nuclear polyhedrosis of the silkworm, Bombyx mori.

Comparative infectivity and virus neutralization studies on occluded and nonoccluded viruses of Bombyx mori nuclear polyhedrosis revealed that the infectious unit causing peroral infection differed from that causing hemocoelic infection. There were functional differences between the occluded (mainly virons with envelopes) and the nonoccluded virus (mainly virions without envelopes) preparations. The peroral infection was largely due to the virion with an envelope (peroral infectious unit), and the hemocoelic infection was due largely to the virion without an envelope (hemocoelic infectious unit). The apparent change of the virions with envelope to those without envelopes was detected as a slight increase in hemocoelic infectivity when the occluded virus was diluted and incubated at 4°C for more than 6 days.     

Physical characteristics of the drumming of Meconema thalassinum

The drumming sound of Meconema thalassinum (De Geer) (Orthoptera: Tettigoniidae: Meconematinae) was studied in a North American population. Males of Meconema produce sound by tapping one of their hind feet on the substrate. Each phrase consists of a variable number (0–5) of short impact trains (mean, 5.6 foot impacts per train), followed by 1–5 long impact trains (mean, 34.4 impacts). The most common type of phrase consists of 3 short and 2 long trains.Foot impacts are produced at rates between 29 and 62/sec in the range 21.0–31.2°C. The foot-impact frequency follows a linear relationship with temperature; a similar relationship holds for the impact-train repetition rate, expressed as the reciprocal of the silent intervals between trains. The number of impacts per train and the overall phrase structure are essentially temperature-independent.The drumming of Meconema is compared with the sounds produced by other Orthoptera, in terms of temperature dependence and of complexity of the phrase structure.     

Parasitism capacity and searching efficiency of Diaeretiella rapae parasitizing Brevicoryne brassicae on susceptible and resistant canola cultivars

Different cultivars of aplant species can affect the foraging and efficiency of natural enemies, both directly through physical and biochemical properties or indirectly through the herbivore's diet. In this study, the parasitism capacity and functional response of Diaeretiella rapae McIntosh were determined on the cabbage aphid, Brevicoryne brassicae (L.) reared on susceptible (Opera) and resistant (Okapi) canola cultivars under laboratory conditions at 25?±?1?°C, 60?±?5% RH and a16:8?h L:D photoperiod. The parasitoid exhibited Type II and Type III functional responses on the resistant and susceptible cultivars, respectively. The estimated value of searching efficiency (a) was 0.1637?±?0.1095?h^?1 on the resistant cultivar whereas its value was dependent on host density on the susceptible cultivar. The handling times (T_h) on the susceptible and resistant canola cultivars were 0.108?±?0.040 and 0.320?±?0.048?h, respectively. The net parasitism rate (C₀) of the parasitoid wasp varied from 128.09 hosts per parasitoid lifetime on the susceptible to 71.01hosts on the resistant canola cultivar. The transformation rate from host population to parasitoid offspring (Qp) was equal to 1 on both cultivars (C₀?=?R₀). The finite parasitism rate (ω) on the susceptible cultivar (0.819 hosts per parasitoid per day) was significantly higher than that on the resistant one (0.578 hosts per parasitoid per day). In conclusion, canola cultivars affected the performance of D. rapae in controlled small-scale laboratory experiments and compared with the susceptible cultivar, the resistant one had anadverseeffect on the efficiency of the parasitoid.