Insects, as a group, are known to be infected by a range of both DNA and RNA viruses (18). Amongst these, the Baculoviridae, Poxviridae, Parvoviridae, and Reoviridae cause most of the known viral diseases in Lepidoptera. The two genera within the Baculoviridae, Granulovirus (GV) and Nucleopolyhedrovirus (NPV), have attracted the most interest as potential agents for use in the suppression of forest insect pest populations because (ⅰ) they are restricted to insects, (ⅱ) they tend to be host specific, and (ⅲ) many are known to cause epizootics within host populations. For example, population crashes due to NPV epidemics occur in many species of sawflies (Hymenoptera: Symphyta). Here, NPV infection is density dependent, and these insects can be particularly susceptible to the communication of the disease as many are communal and feed openly on foliage (Table 1). Attempts to use NPVs to suppress sawfly populations have usually met with success (7, 40). Similarly, in forest Lepidoptera, successful use of NPVs in pest suppression has been achieved with insects that feed openly on foliage and where NPV epizootics occur in a density-dependent fashion (Table 2). Examples where some degree of population suppression has been achieved through the application of NPVs are the Douglas fir tussock moth (Orgyia pseudootsugata), the white-marked tussock moth (O. leucostigma), and the gypsy moth (Lymantria dispar) (7, 33).
Table 2. Application and efficacy of nucleopolyhedroviruses against lepidopteran larvae (7)
Baculoviruses are double-stranded DNA viruses with circular genomes that range in size from 84 kilobases (kb) to over 160 kb (26). Genes are expressed in a transcriptional cascade where each successive phase depends on the successful expression of genes during the previous phase (6, 14, 15). Until recently, all fully sequenced baculovirus genomes, with the exception of the Culex nigripalpus (Diptera) NPV (CuniNPV) (1), had been from Lepidoptera (17). Sawfly NPVs (NeleNPV from Neodiprion lecontei (22), NeseNPV from N. sertifer (12), and NeabNPV from N. abietis (8), all Diprionidae] have the smallest genomes (≈82-86 kb and 89~93 open reading frames (ORFs)) and lowest G+C content (≈34%) of any published baculovirus genome. Baculoviruses typically have a conserved gene order within genera where Lepidoptera-infecting GVs show less parity to lepiodpteran NPVs than to other GVs. Hymenoptera-infecting NPVs, however, show little parity with lepidopteran NPVs or GVs, and even less with the dipteran baculovirus CuniNPV (22). Parity between sawfly baculoviruses, however, implies that there is a strong evolutionary relationship between them (8, 21). Sawfly baculoviruses clearly represent a distinct clade that diverged more recently than Diptera-infecting CuniNPV, but before Lepidoptera-infecting NPVs and GVs (12, 22).
Baculoviruses are transmitted through ingestion by a suitable host larva. In NPVs, virions are ingested as inclusions within polyhedrin protein occlusion bodies (OBs). In the alkaline environment of the larval insect midgut lumen (pH > 10), OBs dissolve, thereby releasing virions to infect midgut epithelial cells (13, 19, 11). In lepidopteran NPVs, the virus goes through an initial phase of replication in the midgut epithelium and produces singly enveloped virions that bud out (budded virus or BV) of the midgut cells to initiate the second round of infection that takes place in cells and tissues within the host hemocoel, such as hemocytes, tracheal cells, and fat body (39, 9). By the time these other cells and tissues have been infected, the infected midgut cells have been replaced by healthy cells so that the midgut becomes cured of infection. At later times following infection, the virions that are produced in the nuclei of fat body and other cells within the hemocoel are enveloped and occluded into OBs. The virions in OBs are referred to as occlusion-derived virions or ODVs. Infection of the midgut with subsequent curing as other tissues become infected results in continued feeding by affected larvae and death may not occur until 2-3 weeks after initial infection of the midgut. When lepidopteran larvae die from NPV infection, they often consist of little more than exoskeletons filled with OBs (109 to 1010 OBs per late instar larva) (39). The viral expression of cathepsin and chitinase ensures the release of the OBs in the environment following the disintegration of the larval cadavers (20, 16).
Sawfly NPVs, on the other hand, only infect the midgut epithelium so that, following initial viral replicative cycles, infected midgut cells containing OBs, are sloughed off into the frass and out of the body where they can infect other host insects (10). Death normally occurs within 1-2 weeks but, during that time, the host is producing infective units of the disease. It is likely that the continuous produc- tion and excretion of OBs from the infected host, along with the gregarious feeding habits of sawfly larvae, allows for sawfly NPV application rates, in control programs, that are two to three orders of magnitude lower than those of lepidopteran NPVs (Table 1 and Table 2).
The isolate of NeabNPV used to develop and register the commercial product AbietivTM (24, 25) was first collected from a population of balsam fir sawfly larvae south of Corner Brook (lat. 48° 57'N, long. 57° 57'W) NL in 1997 (31). Initial amplification of NeabNPV was done in the laboratory. As there are no in vitro systems for sawfly NPV production or artificial diets for sawfly rearing, all balsam fir sawfly larval rearings were done on clean, fresh balsam fir foliage that was artificially contaminated with NeabNPV by misting the foliage with an aqueous suspension of NeabNPV (106 OBs/mL). In July 1999, the total laboratory production of NeabNPV (3.3×109 OBs) was applied by helicopter, in 50 L of 20% aqueous molasses, on 2-3 ha of balsam fir sawflyinfested balsam fir forest. This field production resulted in sufficient NeabNPV to treat 1800 ha at a rate of 1×109 OBs/ha. Since 2000, NeabNPV field production has been done using fixed-wing aircraft. Each autumn, balsam fir stands across Newfoundland, but particularly in the west of the island, are routinely sampled by the Newfoundland and Labrador Department of Natural Resources to determine balsam fir sawfly egg densities to predict defoliation in the following year. Using these data, stands with egg densities averaging between 500–2000 eggs per 45-cm balsam fir branch were selected for NeabNPV production. An application rate of 3×109 OBs/ha in 2.5 L 20% aqueous molasses has been targeted against balsam fir sawfly larvae at roughly the third instar. Each year, from 2000 through 2006, 20-75 ha of forest were treated for NeabNPV production. Beginning 1 week after NeabNPV production applications, and continuing for the next 2-3 weeks, trees in the treated areas were beaten with garden rakes and falling larvae were collected on tarpaulins placed under the trees. The collected materials were then transferred to 40-kg sugar bags, additional balsam fir foliage and NeabNPV from a hand atomizer were added, and the bags were clipped shut and placed in a building at the Canadian Forest Service (CFS), Pasadena Field Station in Pasadena, NL. Insects were kept in the sugar bags until all feeding had ceased, at which point, the balsam fir branches were removed and the remaining contents transferred to 20-kg brown paper bags, which were then stapled shut. As the contents were quite dry, the dead larvae, needles, and other materials could be stored at ambient laboratory conditions (18-22℃). Dead balsam fir larvae were separated from needles and other debris using a blower-box developed by Benoit Morin especially for this purpose (Fig. 1. A and B). Collected larvae were stored frozen (–20℃) in 50-mL centrifuge tubes (Fig. 1. C) until Neab-NPV OBs were purified and counted (Moreau et al. 2005). Suspensions of 4×109 OBs/mL of NeabNPV were divided into aliquots of 40 mL in 50-mL centrifuge tubes (Fig. 1D). Field efficacy trials primarily employed the Cessna 188 aircraft (Table 3, Fig. 1E), which has a load capacity of 400 L. For ease of appli-cation, the formulation is adapted so that one entire tube (40 mL×4×109 OBs/mL=1.6×1011OBs) is added to the hopper of the airplane (400 L 20% aqueous molasses) and, applied at a rate of 2.5 L mix/ha, yields an application rate of 1×109 NeabNPV OBs/ha over a total area of 160 ha.
Figure 1. Neab NPV production. A: Dead balsam fir sawfly larvae and needles are placed in 20-kg grocery bags (GB) for storage. The blower consists of a wooden box with bathroom exhaust fans (BF) on opposite sides. A light switch (LS) turns the blowers on or off. Needles, along with dead insects, are scooped up in a beaker (B) that has a screen on the bottom and is open at the top. The beaker is then placed in the opening (arrow) of the plexiglass funnel (PF). Because the needles are lighter than the dead balsam fir sawfly larvae, the needles blow out of the funnel, through the dryer duct (DD) and are collected in a plastic tub (PT) and discarded. When the blower is turned off, the dead insects fall back into the beaker and are placed in a separate container (not shown) for further processing. B: The staged removal of the needles and capture of the dead balsam fir sawfly larvae moving from bottom to top. The sample is progressively cleaned of balsam fir needles and other unwanted debris until mostly NeabNPV-killed balsam fir sawfly larvae remain. C: Clean larvae are stored frozen in 50-mL conical centrifuge tubes until NeabNPV is purified from them. D: Abietiv ready for application. There is sufficient NeabNPV in each of these 50-mL centrifuge tubes to treat 160 ha of balsam fir sawfly-infested forest at a rate of 1×109 NeabNPV OBs/ha in 2.5 L 20% molasses/ha. E: Forest Protection Limited Cessna 188 loaded with Abietiv and taxiing for take-off at Deer Lake Airport, NL. F: Printout of a spray operation carried out over an irregular-shaped block in western Newfoundland in 2001. The lines within the block indicate where the spray booms were on. Gaps in the lines are either water bodies, cutovers or other areas where there are few or no trees.
Table 3. Aerial field trial applications of NeabNPV against the balsam fir sawfly in western Newfoundland