**4. Experimental designs for evaluating preventative treatments**

When evaluating preventative treatments one of three experimental designs is generally used. Each has its own advantages and disadvantages. Laboratory assays require trap‐ ping and/or rearing of live bark beetles for inclusion in experiments. Captured individu‐ als are immediately transported to the laboratory, identified and sorted. Damaged (e.g., loss of any appendages), weakened, or beetles not assayed within 48 h after collection should be discarded. Generally, serial dilutions of each insecticide are prepared, and tox‐ icity is determined in filter paper or topic assays [7]. The life-table method is used to es‐ timate the survival probability of test subjects to different doses of each insecticide [7]. Filter paper assays more closely approximate conditions under which toxicants are en‐ countered by bark beetles during host colonization, especially for products other than contact insecticides [7], but both methods ignore important environmental factors (e.g., temperature, humidity and sunlight) and host tree factors (e.g., architecture) that influ‐ ence efficacy. However, results are rapidly obtained with limited risk and loss of scientif‐ ic infrastructure compared to field studies.

A second design involves field assays in which insecticides are applied to an experimen‐ tal population of ~25−35 uninfested trees [8]. Trees are often baited with a bark beetle species-specific attractant to increase beetle "pressure" and challenge the treatment fol‐ lowing application. Efficacy is based on tree mortality and established statistical parame‐ ters [8]. This design is accepted as the standard for evaluating preventative treatments for tree protection in the western U.S., and provides a very conservative test of efficacy [9]. However, it is laborious, time-consuming (i.e., generally efficacy is observed for at least two field seasons) and expensive. Experimental trees may be lost to woodcutting or wildfire, and ≥60% of the untreated control trees must die from bark beetle attack to demonstrate that significant bark beetle pressure exists in the area or the experiment fails and results are inconclusive [8]. Some have argued that the design is perhaps too conser‐ vative as under natural conditions aggregation pheromone components would not be re‐ leased for such extended periods of time as often occurs with baiting. Finally, bark beetles may initiate undesirable infestations near experimental trees as a result of baiting, which may be unacceptable under some circumstances.

**Figure 4.** A common method of protecting trees from bark beetle attack is to saturate all surfaces of the tree bole using a ground-based sprayer at high pressure. Photos: C.J. Fettig, Pacific Southwest Research Station, USDA Forest

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Bole sprays are typically applied in late spring prior to initiation of the adult flight period for the target bark beetle species. However, bole sprays require transporting sprayers and other large equipment, which can be problematic in high-elevation forests where snow drifts and poor road conditions often limit access. Additionally, many recreation sites (e.g., camp‐ grounds) where bole sprays are frequently applied occur near intermittent or ephemeral streams that are associated with spring runoff, limiting applications in late spring due to re‐ strictions concerning the use of no-spray buffers to protect non-target aquatic organisms. For these and other reasons, researchers are evaluating alternative timings of bole sprays

Carbaryl is an acetylcholinesterase inhibitor that prevents the cholinesterase enzyme from breaking down acetylcholine, increasing both the level and duration of action of the neuro‐ transmitter acetylcholine, which leads to rapid twitching, paralysis and ultimately death. Carbaryl is considered essentially nontoxic to birds, moderately toxic to mammals, fish and amphibians, and highly toxic to honey bees, *Apis mellifera* L., and several aquatic insects [15]. However, carbaryl is reported to pose little or no threat to warm-blooded animals. Several experts report that carbaryl is still the most effective, economically-viable, and ecologicallycompatible insecticide available for protecting individual trees from mortality due to bark beetle attack in the western U.S. [9,16]. Today, carbaryl (e.g., Sevin® SL and Sevin® XLR Plus, among others) is commonly used to protect trees from bark beetle attack, and is the mostextensively studied active ingredient registered for use. Failures in efficacy are rare and typ‐ ically associated with inadequate coverage, improper mixing (e.g., using an alkaline water source with pH >8) [17] or inaccurate mixing resulting in solutions of reduced concentration, improper storage, and/or improper timing (e.g., applying treatments to trees already suc‐

Service.

**5.1. Carbaryl**

and less laborious delivery methods.

cessfully attacked by bark beetles).

The "hanging bolt" assay [10], "small-bolt" assay [11] and similar variants have received limited attention in the western U.S. Typically, insecticides are applied to individual, unin‐ fested trees that are later harvested and cut into bolts for inclusion in laboratory and/or field experiments. Alternatively, freshly-cut bolts may be treated directly in the laboratory. Effica‐ cy is often based on measures of attack density or gallery construction by adult beetles. Compared to [8], these methods allow for rapid acquisition of data; reduced risk of loss to scientific infrasture; and increased probability that a rigorous test will be achieved as bolts are transported to active infestations or brought into the laboratory and exposed to beetles. While these methods account for some host factors (e.g., bark architecture), others such as host defenses and environmental factors are ignored. Furthermore, the hanging bolt and small bolt assays do not provide an estimate of tree mortality, while the effectiveness of any preventative treatment is defined by reductions in tree mortality.
