Peter M. Ebling

Persistence of naturally occurring and genetically modified Choristoneura fumiferana nucleopolyhedroviruses in outdoor aquatic microcosms.

BACKGROUND To assess the persistence of genetically modified and naturally occurring baculoviruses in an aquatic environment, replicate (three) outdoor, aquatic microcosms were spiked with spruce budworm viruses [Ireland strain of Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and the recombinant CfMNPVegt(-)/lacZ(+)] at a rate of 1.86 x 10(10) occlusion bodies (OBs) m(-2) of surface area. The presence of virus in water samples collected at various times after inoculation was determined by PCR amplification of baculoviral DNA extracted from OBs. RESULTS Although UV radiation rapidly degrades baculoviruses under natural conditions, both viruses persisted above the level of detection (>100 OBs 450 microL(-1) of natural pond water) for at least 1 year post-inoculation, with little difference between the viruses in their patterns of persistence. CONCLUSION The present microcosm study suggests that occlusion bodies of baculoviruses can persist in the flocculent layer of natural ponds. On disturbance, OBs could re-enter the main water column and thus be available for transport to new locations. Implications for environmental risk assessment are discussed.

DNA hybridization assay for detection of nucleopolyhedrovirus in whitemarked tussock moth (Orgyia leucostigma) larvae

DNA dot-blot hybridization assays utilizing a horseradish peroxidase-labelled whole genomic DNA probe and enhanced chemiluminescence were conducted to quantify detection thresholds of nucleopolyhedrovirus (NPV) in whitemarked tussock moth (Orgyia leucostigma) larvae. The minimum detection thresholds for an aqueous suspension of occlusion bodies (OBs), OBs added to macerates of non-infected larvae and OBs in macerates of diseased larvae were 7.8 x 10(3), 7.8 x 10(3), and 1.5 x 10(3) OBs, respectively. Purified viral DNA was detected at a concentration of 1.6 x 10(-1) ng in a 20 microliters volume. The presence of pre-occluded viral nucleocapsids and DNA, inherent to infected larvae, improved the detection threshold five-fold compared with OBs alone. Larval tissues did not block the detection system utilized, nor did they bind non-specifically to the probe. Detection thresholds, upon sequential hybridization of the same membrane, on average deteriorated two-fold between the first and second hybridization and an additional six-fold between the second and third hybridization. NPV infection was detected two days post-inoculation (pi) in about one-third of the larvae examined and in almost all larvae three days pi. Microscopic analysis of stained larval smears missed NPV infection in almost all larvae two days pi and about two-thirds of the larvae three days pi. Results from the two methods of analysis were not comparable until four days pi. The detection system utilized is a reliable, efficient and simple method for the early detection of NPV infection in large numbers of larvae and may be used for further studies quantifying the role of this baculovirus in the ecology of whitemarked tussock moth populations.

Host-Range Testing of a Mixture of Two Nucleopolyhedroviruses of Choristoneura fumiferana (Lepidoptera: Tortricidae)

Abstract The host range of a mixture of Choristoneura fumiferana (Clemens) nucleopolyhedroviruses (CfMNPV and CfDefNPV) was investigated using a per os bioassay of larvae of 29 species of Lepidoptera and adult males of Megachile rotundata (F.) (Hymenoptera: Megachilidae). Using a whole-genomic DNA probe, positive results were obtained in 8 of 10 Tortricidae: Archips cerasivorana (Fitch), Choristoneura fractivittana (Clemens), C. fumiferana, Choristoneura occidentalis Freeman, Choristoneura pinus pinus Freeman, Choristoneura rosaceana (Harris), Clepsispersicana (Fitch), and Cydia pomonella (L.); one Crambidae: Ostrinia nubilalis (Hübner); one arctiine Erebidae: Estigmene acrea (Drury); and two Noctuidae: Oligia illocata (Walker) and Pyrrhia exprimens (Walker). Mortality rates were highest among C. fumiferana, C occidentalis, C. pinus pinus, A. cerasivorana, and C. pomonella. Sequenced polymerase chain reaction (PCR) amplicons from infected individuals from several species confirmed that the primer sets amplified the target viruses. CfMNPV was consistently found in virus-fed C. fumiferana; whereas, CfDefNPV was present only occasionally. The presence of CfMNPV and CfDefNPV in A. cerasivorana was confirmed by PCR and DNA sequencing. Significant treatment-mortality rates were induced in the noctuids P. exprimens and Acronicta impleta Walker; PCR determined that both viruses were present in treated P. exprimens but only CfMNPV was present in A. impleta. No virus was detected in M. rotundata.

Effects of diapause duration on postdiapause performance of spruce budworm (Lepidoptera: Tortricidae) during mass rearing on artificial diet

The spruce budworm, Choristoneura fumiferana (Clem.), has an obligatory winter dormancy period that lasts up to 10 months in the field. In the Great Lakes Forestry Centre rearing facility, neonate larvae spin hibernacula in cheesecloth, which is then stored at 2 °C for between 20 and 30 weeks. Although dormancy survival and synchrony of postemergence development are highest when larvae are stored in the cold for 16–35 weeks, it is not known how cold-storage duration affects spruce budworm performance once diapause has been completed. We exposed approximately 9250 second-instar larvae (belonging to three rearing cohorts) to 2 °C for 16, 19, 22, 25, 28, 31, 34, or 37 weeks and monitored various postdiapause performance variables. Increasing cold storage from 16 to 25 weeks or more resulted in small (approximately 10%) increases in dormancy survival and larval development rates (from second instar to pupation), a larger (up to 23%) increase in pupal mass and realized fecundity (number of eggs laid per female...

PATHOGENICITY OF CHORISTONEURA FUMIFERANA NUCLEOPOLYHEDROVIRUS PROPAGATED IN VITRO AT DIFFERENT INCUBATION TEMPERATURES

SummaryTo optimize the in vitro production of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) as a potential microbial pest control agent, the pathogenicity of occlusion bodies (OBs) produced in two cell lines at three incubation temperatures was determined by bioassay. A plaque-purified isolate of CfMNPV was amplified in permissive C. fumiferana cell lines, FPMI-CF-203 and FPMI-CF-2C1, and incubated at 22, 24, and 28° C. Occlusion bodies propagated in FPMI-CF-203 cells at 28° C were significantly larger (17.5 μm3) and more pathogenic (LD50=27; LD95=185, where LD50 and LD95 are doses required to kill 50 and 95% of the test larvae, respectively) than those produced in either of the cell lines at any of the incubation temperatures tested. Increased temperatures yielded larger OBs from both cell lines. The pathogenicity of OBs propagated in the FPMI-CF-203 cell line increased with incubation temperature, whereas that of OBs produced in FPMI-CF-2C1 cells decreased. Comparison of the pathogenicity of OBs, whether naturally occurring or genetically modified, should be standardized by cell line and incubation temperature used for propagation. Production efficiency decreased with increasing incubation temperature for each cell line. Lower incubation temperatures used for propagation, and standardization of the titer of viral inoculum, should be further investigated to determine the economic feasibility of the in vitro production of CfMNPV as a microbial pest control agent.