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Weekly UpdatesTo determine the maximum weight loss of larvae, five ALB larvae were held for 64 hours at a vacuum pressure of 20 mmHg at 20[degrees]C. Ten EAB larvae were held in a drying oven (Blue M Electric Co., single-wall gravity convection laboratory oven) at 100[degrees]C for 94 hours. Five pupae and 11 eggs of ALB were also exposed to a vacuum pressure of 20 mmHg at 20[degrees]C for 20 hours to assess response.
After the tests, larvae were considered dead if they did not move when probed within 3 hours of removal from treatment. ALB larvae that survived vacuum treatment were returned to the artificial diet on which they had been reared (Keena 2005), and their survival and development were monitored. Pupae were considered dead when no movement was observed within 3 hours, and they did not complete development to adults within 3 weeks after being held at 25[degrees]C and 60 percent RH. After vacuum treatment, the condition of eggs was observed, and they were held at 25[degrees]C in a high-humidity environment (as described in Keena 2005) for 3 weeks to determine viability and hatching success.
Vacuum lethal time at different test temperatures and pressures
The relationship between temperature and vacuum lethal time was assessed. Twenty-five ALB larvae were exposed to 20 mmHg pressure at 30[degrees]C for 4 to 6 hours and 40 EAB larvae were exposed to 20 mmHg at -10[degrees]C for 24 or 36 hours to investigate desiccation of larvae at these temperatures. Fifteen ALB larvae were held at each of two additional vacuum pressures, 10 and 75 mmHg, at 20[degrees]C to evaluate the relationship between vacuum pressure and lethal vacuum time.
Vacuum treatment of larvae inserted into wood
Thirty ALB larvae and 20 EAB larvae were inserted into pieces of wood (dimensions given below) with varying MC to determine the relationship between desiccation rate and MC. Larvae were inserted into holes (4 cm deep) that were drilled in test wood pieces. Holes were 1 cm or 0.5 cm in diameter for ALB and EAB, respectively. The holes were securely plugged after inserting the larvae with pieces of wood dowel. Vacuum treatment was applied within 3 hours of placing larvae in the test wood pieces. Immediately following treatment, larvae were removed from the holes, placed on moist filter paper in petri dishes, and maintained at room temperature. Larvae were considered dead if they did not move when probed within 3 hours of removal.
Ten ALB larvae were placed in separate wood test pieces at each of three wood moisture levels: (1) commercially available Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) lumber, 2.5 cm wide by 10 cm long by 2.5 cm thick at 21.6 percent MC, (2) commercially available Douglas-fir lumber 10 cm wide by 10 cm long by 2.5 cm thick at 31.4 percent MC, and (3) freshly cut Norway maple (Acer platanoides L.) 5 cm thick by 10 cm diameter at 89.4 percent MC. Half of the test wood pieces of each type with larvae inserted were held at 20 mmHg and 20[degrees]C and the other half at 10 mmHg and 30[degrees]C to determine the effect of wood MC on vacuum lethal time.
Twenty EAB larvae were placed in separate wood test pieces cut from commercially available Douglas-fir 2.5 cm wide by 10 cm long by 2.5 cm thick. The MC of the wood was 16.6 percent. The test wood pieces were held at 20 mm Hg and 20[degrees]C.
Data analysis
Lethal percentage weight loss for ALB and EAB held at 20 mmHg and 20[degrees]C was determined by probit analysis (Robertson and Preisler 1992) with Polo Plus (LeOra Software, Berkeley, California). Desiccation mortality response lines for the two beetle species were compared by a Chi-square test (PROC FREQ, SAS Institute 2002-2003). Desiccation rates at 20 mmHg and 20[degrees]C of ALB and EAB were compared using a t-test (PROC t-test, SAS Institute 2002-2003). For each species, desiccation rates for larvae held at two different temperatures were compared using a t-test. Differences in desiccation rates of ALB held at three different vacuum levels were compared using analysis of variance (ANOVA, PROC GLM, SAS Institute 2002-2003). The desiccation rates of ALB larvae and pupae were compared using a t-test. For each species, the desiccation rates of larvae exposed directly to vacuum at 20 mmHg and 20[degrees]C were compared to larvae inserted into wood and exposed to the same vacuum and temperature conditions by a t-test (PROC t-test, SAS Institute 2002-2003). Desiccation rates of ALB larvae inserted into wood were compared for the three different wood MC levels by ANOVA followed by the Ryan-Einot-Gabriel-Welsh (REGW) multiple comparison procedure (PROC GLM, SAS Institute 2002-2003).
Results and discussion
Effectiveness of vacuum treatment and lethal percentage weight loss of larvae
The desiccation mortality response line (Fig. 2, solid line, intercept -23.2 [+ or -] 5.3, slope 15.6 [+ or -] 3.6, probit mortality vs. log % weight loss) for ALB predicts that 50 percent of the larvae will be dead after 31.2 percent weight loss and that virtually all (probit 9) the larvae will be dead after 56.4 percent weight loss. The desiccation mortality response line (Fig. 2, dashed line, intercept -4.5 [+ or -] 3.4, slope 9.4 [+ or -] 2.3, probit mortality vs. log % weight loss) for EAB predicts that 50 percent of the larvae will be dead after 34.5 percent weight loss and that virtually all (probit 9) the larvae will be dead after 91.9 percent weight loss. The desiccation mortality curve for EAB does not provide as good a fit to the data so the estimates for the lethal percentage weight loss, and predicted mortality based on the probit mortality curves is not as accurate. The response lines for the two beetle species are not significantly different from each other ([chi square] = 3.64, df = 2, p = 0.162).
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These results demonstrate that both ALB and EAB larvae can be killed by low-pressure vacuum treatment through evaporative removal of body water. ALB larvae died after losing as little as 26 percent total body weight, and all larvae in the trials that lost [greater than or equal to]35 percent weight died (Fig. 2). EAB larvae died after losing as little as 28 percent total body weight and all larvae that lost >39 percent weight died (Fig. 2). Similar percentage dehydration is tolerated by other beetles; certain tenebrionids tolerated >50 percent, chrysomelids survived up to 46 percent water loss, and two other cerambycids survived 35 to 40 percent weight loss (Gehrken and Somme 1994, Chen et al. 2007).
The desiccation curves (mean cumulative percentage weight loss vs. time) for both ALB (Fig. 3) and EAB (Fig. 4) larvae were linear during the first part of the desiccation process. The desiccation rate for ALB decreased after about 30 hours at 20 mmHg and 20[degrees]C as weight loss reached approximately 50 percent and larvae approached complete desiccation. Complete desiccation for ALB larvae required about 50 hours at 20 mmHg and 20[degrees]C. The maximum weight loss under 20 mmHg at 20[degrees]C for ALB larvae ranged from 60 percent to 67 percent with an average of 62.8 percent. The maximum weight loss for EAB larvae was similar and ranged from 58.5 percent to 63.1 percent with an average of 60.9 percent. Terrestrial arthropods usually contain 65 to 75 percent water by weight (Hadley 1994). Linear desiccation rates also have been observed for some tenebrionid beetles (Renault and Coray 2004).
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The desiccation rate for ALB larvae was negatively correlated with initial larval weight (Fig. 5, desiccation rate = -1.5 x Initial weight + 5.9, [r.sup.2] = 0.28). Similarly, the desiccation rate for EAB larvae was negatively correlated with initial larval weight (Fig. 6, desiccation rate = -17.7 x Initial weight + 3.78, [r.sup.2] = 0.22). ALB larvae desiccated significantly faster (3.35 % weight loss per hour, t = -5.57, df = 105, p < 0.0001) than EAB larvae (2.39 % weight loss per hour) under 20 mmHg at 20[degrees]C.
It took longer to kill EAB larvae than ALB larvae under vacuum, despite the fact that EAB larvae are smaller than ALB larvae and thus have a higher surface area to volume ratio. It is possible that ALB and EAB differ in cuticular composition and/or respiratory rate. Both factors are related to transpiration and desiccation in insects (Addo-Bediako et al. 2001). The EAB used in these experiments were prepupal larvae that had completed feeding, were overwintering, and were held in cold storage. On the other hand, the ALBs used in these experiments were laboratory-reared on artificial diet at room temperature and had not yet reached the pupal stage or completed feeding. Overwintering insects have adaptations to cope with the potential for ice formation in their body fluids, including the production of sugars (trehalose) and antifreeze (glycerol) in the haemolymph (Lee Jr. 1989). Production of osmolytes not only protects against freezing, but also decreases the gradient of water vapor pressure and thus provides some protection against desiccation (Kaersgaard et al. 2004). Moreover, the presence of trehalose protects membranes from morphological damage during drying allowing organisms to survive high levels of desiccation, a condition known as anhydrobiosis (Crowe et al. 1992). Because the EAB larvae used in these experiments were collected from their overwintering sites, they may have had a higher concentration of trehalose and osmolytes than the laboratory-reared ALB and thus may have been somewhat protected from desiccation through a reduced desiccation rate and an increased ability to survive higher levels of desiccation.
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Vacuum lethal time at different test temperatures and pressures
Desiccation rate was directly related to temperature during vacuum treatment. Desiccation rate increased significantly at higher temperatures (t = 9.86, df = 55, p < 0.0001). For example, ALB larvae lost weight twice as fast at 30[degrees]C (6.17 % weight loss per hour) compared to 20[degrees]C (3.35 % weight loss per hour) (Table 1). Similarly, for A. planipenis larvae, the desiccation rate at 20[degrees]C (2.39 % weight loss per hour) was significantly higher than at -10[degrees]C (0.13 % weight loss per hour) (t = 20.30, df = 113,p < 0.0001). After 36 hours under 20 mmHg at -10[degrees]C all of the EAB larvae were still alive. Mbata and Phillips (2001) also found that higher temperatures resulted in significant reductions in vacuum lethal time for stored-product insects. As temperature increases so does respiration which is one of the avenues through which water is lost in insects (Hadley 1994). At below-freezing temperatures, factors involved in cold tolerance may have also contributed to increased lethal time and protection from desiccation. Regulation of internal osmotic pressure by means of sugars protects against freezing (Lee Jr. 1989) and desiccation (Kaersgaard et al. 2004). Furthermore, when insects are exposed to below-freezing temperatures, the haemolymph becomes concentrated by formation of ice until the vapor pressure of the liquid fraction equals that of ice at the same temperature. The haemolymph of frozen insects in a closed frozen hibernaculum is therefore in vapor pressure equilibrium with the air in the frozen hibemaculum, thus these insects do not lose water during winter (Lundheim and Zachariassen 1993). The slower desiccation rate at below freezing temperatures was not likely due to drier conditions because relative humidity in the vacuum chamber was similar at 20[degrees]C (2.7%) and -10[degrees]C (2%).
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