Evaluation of vacuum technology to kill larvae of the Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae), and the emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae), in wood.

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.

[FIGURE 6 OMITTED]

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%).

For ALB there were significant differences between the desiccation rates at the three vacuum levels tested. Desiccation rate increased significantly under greater vacuum (i.e., lower pressure mmHg) (F = 87.36; df = 2, 59; p < 0.0001); the desiccation rate was 4.82, 3.35, and 0.64 percent weight loss per hour under 10, 20, and 30 mmHg, respectively, at 20[degrees]C (Table 1). Similarly, Mbata et al. (2004) found that stored product insects died more rapidly under greater vacuum (i.e., lower pressure mmHg).

Six ALB pupae were tested under 20 mmHg at 20[degrees]C. Five of them died and one was badly damaged by the test. The pupae lost 26 to 46 percent total body weight. The desiccation rate for ALB pupae (1.88 % weight loss per hour) was significantly slower than for larvae (3.35 % weight loss per hour, t = -3.07; df = 35; p = 0.004). Similarly, Mbata and Phillips (2001) found that the pupae of stored-product insects were more tolerant to low pressure than larvae.

Eleven A. glabaripennis eggs held individually in the wells of a 24-well plate were exposed to 20 mmHg at 20[degrees]C. Two tests were conducted; one using five eggs and the other using six eggs. The eggs all appeared to have collapsed after exposure to the same vacuum treatment as the pupae; however, one egg did hatch after they were returned to a high humidity environment.

Vacuum treatment of larvae inserted into wood with different MCs

Wood MC must be taken into account when developing a treatment scheme for SWPM. Relative humidity inside the oven or container used for the vacuum treatment will increase when wetwood is placed inside the chamber. For instance, relative humidity in the vacuum chamber was 2.7 percent when only larvae were placed in the chamber, while it was 28.1 percent when wood blocks containing larvae were placed in the chamber under the same temperature (20[degrees]C) and vacuum pressure (20 mmHg) conditions.

The desiccation rate of A. glabraipennis larvae subjected directly to 20 mmHg at 20[degrees]C (average 3.35 percent weight loss per hour) was significantly higher than for larvae inserted into wood (average for all wood MC levels 1.42 percent weight loss per hour, t = 9.86, df= 55, p < 0.0001). For both vacuum levels tested, desiccation rates of ALB larvae inserted into wood were higher when MC of the wood was lower. At 20 mmHg and 20[degrees]C, ALB larvae inserted into wood with 21.6 percent MC desiccated more rapidly (0.94 [+ or -]0.17 percent weight loss per hour) than larvae inserted into wood with 31.4 or 89.4 percent MC (0.41 [+ or -] 0.06 and 0.23 [+ or -] 0.06 percent weight loss per hour, respectively; F = 11.01, df = 2, 14, p = 0.0019). Similarly, at 10 mmHg and 30[degrees]C, ALB larvae inserted into wood with 21.6 percent MC desiccated more rapidly (2.02 [+ or -] 0.39 percent weight loss per hour) than larvae inserted into wood with 31.4 or 89.4 percent MC (0.61 [+ or -] 0.18 and 0.34 [+ or ] 0.20 percent weight loss per hour, respectively; F = 15.72, df= 2.14, p = 0.0004). Stenocorus lineatus Oliv., sawyer beetles (Monochamus sp.), and pine wood nematodes (Bursaphelenchus xylophilus) all were killed after 24 hours at 20 mmHg at 20[degrees]C when placed in wood with >30% MC (Chen et al. 2007). ALB larvae placed in the test wood pieces at 21.6 percent MC, lost weight (0.94 % weight loss per hour) but at a slower rate than larvae that had been directly exposed (3.35 % weight loss per hour) to the same vacuum treatment (20 mmHg vacuum pressure at 20[degrees]C). Approximately 50 percent of the ALB larvae held in wood under these conditions had lost more than 40 percent body moisture after about 20 hours and were dead. ALB larvae in the wood that had 89.4 percent MC lost weight even more slowly. The results for EAB larvae were very similar to those for ALB. The desiccation rate of EAB larvae directly exposed to 20 mmHg at 20[degrees]C (2.39 % weight loss per hour) was significantly higher than for larvae inserted into wood (0.94 % weight loss per hour, t = 9.14, df= 93,p < 0.0001).

Vacuum treatment can kill ALB and EAB larvae even when inserted into wood; however, lethal time increases with increasing wood MC (Table 1).

The work reported here is a critical step in evaluating the usefulness of vacuum treatment for killing important nonnative wood-dwelling pests that can be transported in SWPM. Further work to determine the exact treatment conditions for wood material in a commercial facility will be necessary if this method is to be considered for adoption as part of ISPM-15 used by the U.S. and its trading partners.

Conclusions

ALB and EAB larvae die under low pressure vacuum due to desiccation. A glabripennis pupae and eggs were also susceptible to desiccation under low pressure vacuum. The lethal percentage total body weight loss for both ALB and EAB larvae was determined to be around 30 to 40 percent, and the percentage mortality response curves were not significantly different from each other. The desiccation rate for both ALB and EAB larvae under vacuum is constant before death, but ALB larvae had 1.4 times higher desiccation rate than EAB larvae under the same vacuum and temperature conditions of 20 mmHg at 20[degrees]C. The desiccation rate was positively correlated with the temperature; at higher temperatures larvae lost weight faster than at lower temperatures. Vacuum pressure also affected the desiccation rate; at lower pressures the desiccation rate was higher. When the relative humidity inside the vacuum container was higher, the desiccation rate decreased, which was the case when larvae were placed in wood with varying MCs.