Patrick R. Hughes

A synchronous peroral technique for the bioassay of insect viruses

Abstract 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.

Myrcene: a precursor of pheromones in Ips beetles

Abstract Males of Ips spp. produced the pheromones ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and/or ipsenol (2-methyl-6-methylene-7-octen-4-ol) when exposed to vapours of myrcene, a monoterpene present in their hosts (Pinus spp.). Ips grandicollis and Ips calligraphus require feeding before metabolizing the myrcene, whereas Ips avulsus and Ips paraconfusus produce some pheromone without prior feeding. Topical treatment with ipsdienol results in ipsenol production in both fed and unfed I. paraconfusus males but only in fed I. gradicollis males. I. calligraphus males, which do not produce ipsenol in nature, did not produce any with the topical treatment regardless of prior conditioning. It is concluded that myrcene can serve as a precursor for these terpene alcohols and suggested that ipsenol is produced by the reduction of ipsdienol. Furthermore, the biosynthesis of these pheromones appears to be under some form of control in certain species, with the stimulus for production occurring upon feeding.

A modified droplet feeding method for rapid assay of Bacillus thuringiensis and baculoviruses in noctuid larvae

Abstract A modification of the droplet feeding method is described which reduces the time and cost of conducting bioassays of pathogens in suitable lepidopterous larvae. The procedure uses a single, large surface for treatment of the insects, and the inoculum for a given dose is administered in a single step by means of a simple applicator. A Petri dish with diet is inverted over the larvae and surrounding droplets of each dose group, permitting the larvae to move directly onto the diet after ingesting the inoculum. This method eliminates the need to transfer individual larvae, and the moist diet prevents evaporation from the droplets during the period of acquisition. After administration of the inoculum, the dishes are removed from the treatment surface, larvae that have not ingested are discarded, and the dishes are covered and placed in an incubator. The mean volume of inoculum ingested per larva was the same for both the applicator and the individual droplet procedures. Results with the applicator technique compare well with other methods, but considerably less time and materials are required to conduct an assay.

Antioxidant responses to simulated acid rain and heavy metal deposition in birch seedlings.

This study measured the responses of different anti-oxidants in 2-year-old birch (Betula pendula Roth) seedlings subjected to simulated acid rain (pH 4.0) and heavy metals (Cu/Ni), applied alone or in combination for 2 months. The applied concentrations of pollutants did not significantly affect seedling biomass or total glutathione levels. Acid rain alone increased superoxide dismutase (SOD) activity both in leaves and roots, while heavy metals alone inhibited SOD activity in roots. Both acid rain and heavy metals applied singly increased ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) activities in leaves but decreased activities in roots. In contrast, acid rain and heavy metal treatments increased glutathione reductase (GR) activity in roots but not in leaves. Spraying birch seedlings with a mixture of acid rain and heavy metals increased SOD, APX and GPX activities in leaves and GR activity in roots. However, the effects of mixed pollutants on enzyme activities usually were less than the summed effects of individual pollutants. Enzyme responses also depended on where pollutants were applied: spraying pollutants onto the shoots initiated higher responses in SOD, APX and GPX than did application to the soil surface, while the opposite was true for GR.

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8-aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.

Pheromones of Dendroctonus: Origin of α-pinene oxidation products present in emergent adults

Abstract Experiments showed that larvae and adults of the bark beetles Dendroctonus terebrans and Dendroctonus frontalis are capable of metabolizing α-pinene, a component of the oleoresin of their host Pinus taeda , to produce large quantities of oxidation products such as trans -verbenol, whereas the pupae do not. The results suggest that the pupae conjugate some form of the terpene molecule with an unknown compound and this conjugate is later metabolized by the young adult to yield the previously identified oxidation products found in emergent beetles. Only adult males of D. frontalis produced large quantities of the ketone verbenone. This compound was not detectable in the hindguts until after the adult maturation period and its production by emergent males could be related to the exposure of the pupae to α-pinene vapours. D. frontalis males are also capable of producing verbenone from α-pinene taken up in the adult stage. It is suggested that the production of verbenone by the males represents a specialization in the evolution of chemical communication in bark beetles. On the basis of this and earlier work, it is considered likely that other terpenes are metabolized in the same manner and that the same or a very similar system of terpene metabolism exists in other Dendroctonus species and closely related genera.

Oxidation products of terpenes identified from Dendroctonus and Ips bark beetles.

Abstract Exposure of adult males and females of Dendroctonus brevicomis and D. frontalis to camphene vapor resulted in oxidation of the terpene to a prominent product, which was identified as 6-hydroxy-camphene (camphenol). Exposure of D. brevicomis adults to myrcene vapor resulted in sex-specific oxidation of the hydrocarbon. A major product in both sexes was identified as 2-methyl-6-methylene-2,7-octadien-1-ol (myrcenol), whereas ipsdienol, a major product in males, was not detected in females. A compound detected in hindguts of feeding males of Ips pini and I. paraconfusus was attributed to the presence of 3-carene in the host ( Pinus spp.) and subsequently identified as 1-methyl-5-(α-hydroxy-isopropyl)-cyclohexa-1,3-diene.

Novel Insect Cell Line Capable of Complex N-Glycosylation and Sialylation of Recombinant Proteins

Paucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1–4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S and the DpN1 cells produced lower concentrations of SeAP than the Tn5B1–4 cells. Less than 5% of the glycans attached to SeAP produced by the Tn5B1–4 cells had complex forms. Glycans attached to SeAP from A7S cells contained 4% hybrid and 8% complex forms. Galactosylated biantennary structures were identified. Glycans attached to SeAP produced by the DpN1 cell line had 6% hybrid and 26% complex forms. Of the complex forms in SeAP from DpN1, 13% were identified as sialylated glycans. The galactosyltransferase activity of the three cell lines was measured and correlated to their ability to produce complex forms. Even though neither novel cell line produced as much recombinant protein as the Tn5B1–4 cells, the glycosylation of SeAP expressed by both cell lines was more complete. These novel cell lines represent interesting alternatives for the production of complex glycosylated proteins utilizing the BEVS.

In vivo enhancement of baculovirus infection by the viral enhancing factor of a granulosis virus of the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae)

Abstract The ability of the viral enhancing factor (VEF) from Trichoplusia ni granulosis virus to enhance the infectivity of Autographa californica multiply enveloped nuclear polyhedrosis virus (AcMNPV) was measured in mid-fourth instar T. ni bioassays. In dose-response bioassays with a constant AcMNPV dose and VEF concentrations varying from 1 to 40 ng/larva, mortality increased linearly with the log dose of VEF from 20.8 to 93.1%. Reciprocal bioassays in which the dose of VEF was constant while that of the virus was varied yielded parallel dose response lines with a calculated potency ratio (LD50 for virus alone/LD50 for virus + VEF) of 3. In addition to increasing mortality, 40 ng of VEF significantly reduced median survival times of LD20 and LD90 doses of AcMNPV by 9.5 and 10.7 hr, respectively. VEF also enhanced AcMNPV in neonates of T. ni. Increased susceptibility of T. ni larvae to T. ni singly enveloped nuclear polyhedrosis virus (a potency ratio of 16 with 40 ng of VEF), and Anticarsia gemmatalis multiply enveloped nuclear polyhedrosis virus (a potency ratio of ca. 10.0 with 40 ng of VEF) was also demonstrated.

Quantitative aspects of nuclear polyhedrosis virus infections in lepidopterous larvae: the dose-survival time relationship

Abstract Dose-survival time relationships are presented for neonate larvae of Heliothis zea inoculated with H. zea singly embedded nuclear polyhedrosis virus (HzSNPV) and Trichoplusia ni inoculated with the 1A clone of Autographa californica multiply embedded NPV, the HOB-mutant of this clone, or T. ni SNPV. For each of these virus-host combinations, plots of median survival time (ST) against logarithm of the inoculum concentration have the shape of an inverted sigmoid curve. The plateau of ST50 values at low doses is interpreted as resulting from the dose received by responding larvae remaining constant while the proportion of treated larvae receiving this dose decreases; evidence is presented that the plateau values at high doses are not produced by saturation of virus receptor sites on the midgut epithelium. A close similarity was noted between the dose-survival time relation of baculoviruses and their hosts on the one hand, and that of vertebrate pathogens and their hosts on the other; for the latter, mathematical modelswith predictive power have been developed.