94 Strom et al.: Evaluating Insecticides for Preventing Southern Pine Beetles process than it does with bole sprays; a consid- eration for developing evaluation criteria. Attack- ing beetles must penetrate the outer bark before they are exposed to a systemic insecticide in the phloem, whereas with bole sprays, beetles may be killed on the outer bole prior to reaching the inner bark. Treated tissues must be contacted, and usually consumed, for the most toxic effect to be realized from systemics, so assessing the number of attacks may provide an incomplete determi- nation of treatment effects. Attack number has not been reduced by even those systemics with the greatest impact against bark beetles (Gros- man et al. 2009; Strom et al. unpublished data), and under certain conditions one would expect the number or density of attacks to increase due to shorter beetle galleries and the consequent increase in acceptable phloem tissue available to subsequent attackers. Strom and Roton (2009) did not evaluate systemic treatments and did not address this difference in treatment activity when proposing guidelines. For systemic treatments, attack number may retain importance, but utili- zation of phloem is also important for assessing toxicity. To address this, researchers of the current study evaluated attack number and the gallery length per attack (GLA) and compared treat- ments using analysis of variance (ANOVA) when sample sizes allowed. Transformation by natural log or square root was used when data histograms and residual plots showed improve- ment toward normality and homogeneous vari- ances; the better of the two transformations for meeting these criteria was selected in each case. This analysis provides a first-step toward pro- ducing thresholds for effectiveness; however, the relatively late exposure of adult beetles during the attack process and the relatively slow effects of some systemic AIs may conspire to reduce their utility under high bark beetle pressures (see Discussion). Readers are cautioned about interpreting ANOVA results as being predictive of resource protection or field (in situ) efficacy. Determination of Imidacloprid Residues Imidacloprid residue concentrations in phloem were assessed with polyclonal enzyme-linked im- munosorbent assay (ELISA) kits (EnviroLogix™, ©2015 International Society of Arboriculture Portland, Maine, U.S.) for untreated and imida- cloprid-treated study trees (Fisher et al. 2009). Upon felling, a representative bolt from each tree was stripped of its phloem, which was then col- lected and kept frozen (-19°C) until preparation of samples for residue determinations. Samples were oven dried at 38°C for at least 48 hours, followed by grinding in a Wiley mill (mesh size 20). A 0.5 g portion of each ground sample was extracted in 10 ml of chromatography grade meth- anol (Burdick and Jackson, Muskegon, Michigan, U.S.) with overnight horizontal agitation. Fol- lowing removal from the agitator, samples were placed upright, sediment was allowed to settle and 1.5 ml from each sample transferred to a 12 mm × 32 mm autosampler vial with a Teflon-lined screw cap (SUN SRI, Rockwood, Tennessee, U.S.). Sam- ples were refrigerated (4°C) until determinations of residue concentrations were made. For ELISA, all kit instructions were followed. Duplicate wells were used for each sample and their mean recorded. The dynamic range of the Envirologix imidacloprid kit is 0.2 to 6.0 ng/ml, so serial di- lutions were made from samples, as needed, until a result within this range was achieved. A Biotek ELx808 plate reader (Biotek, Inc., Winooski, Ver- mont, U.S.) was used to measure absorbance at 450 nm, and results calculated using 4-parameter curve fitting soſtware (Biotek Gen5, Biotek, Inc.). Both matrix-only blanks and imidacloprid spikes (6 µg/g dry weight) were included with each plate to provide an estimate of matrix interferences and to ensure kit performance. The mean value gener- ated from duplicate samples was used for analysis. RESULTS Small bolts from different treatments did not dif- fer in size. The average bolt diameter in SPB field trials was (mean ± 1 SEM) 11.2 ± 0.3 cm, aver- age length was 10.9 ± 0.07 and bark surface area averaged 384.8 ± 9.4 cm2 were 10.9 ± 0.2 cm for diameter, 11.1 ± 0.04 cm for length, and 379.2 + 7.2 cm2 = 0.81, P = 0.53). Because . In lab trials, these values for bark surface area. bolts did not differ in size by treatment, research- ers did not correct for bolt size in evaluations. Two-way analysis of variance was used to com- pare bolt surface area by block and treatment and resulted in no significant differences (field: F4, 13 0.61, P = 0.66; lab: F4, 33 =
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