The Effect of Acclimation to Sublethal Temperature on Subsequent Susceptibility of Sitophilus zeamais Mostchulsky (Coleoptera: Curculionidae) to High Temperatures

Heat treatment is a popular alternative to synthetic pesticides in disinfesting food-processing facilities and empty grain storages. Sitophilus zeamais Mostchulsky is one of the most cosmopolitan and destructive insects found in empty grain storage facilities and processing facilities. The effect of acclimation in S. zeamais adults to sublethal high temperature on their subsequent susceptibility to high temperatures was investigated. S. zeamais adults were acclimated to 36°C for 0 (as a control), 1, 3, and 5 h, and then were exposed at 43, 47, 51, and 55°C for different time intervals respectively. Acclimation to sublethal high temperature significantly reduced subsequent susceptibility of S. zeamais adults to lethal high temperatures of 43, 47, 51, and 55°C, although the mortality of S. zeamais adults significantly increased with increasing exposure time at lethal high temperatures. The mortality of S. zeamais adults with 1, 3, and 5 h of acclimation to 36°C was significantly lower than that of S. zeamais adults without acclimation when exposed to the same lethal high temperatures. The present results suggest that the whole facility should be heated to target lethal high temperature as soon as possible, avoiding decreasing the control effectiveness of heat treatment due to the acclimation in stored product insects to sublethal temperature.


Introduction
The maize weevil, Sitophilus zeamais Mostchulsky (Coleoptera: Curculionidae), is a primary insect pest of cereal grains, particularly in maize and wheat, whose infestation usually starts in the field before harvest and extends in bulk grain and processing facilities [1][2][3]. Control of stored-grain insect pests has ever been primarily achieved by continued applications of methyl bromide and phosphine. However, methyl bromide has been phased out due to its ozone depleting potential according to the Montreal Protocol in the world [4,5]. Currently, control of S. zeamais population is primarily dependent upon intensive use of phosphine [6]. However, its repeated use for decades has disrupted biological control by natural enemies and led to serious concerns about insecticide resistance, environmental contamination, pesticide residue in food, and lethal effects on non-target organisms [7][8][9][10][11].
Recently, the integrated pest management (IPM) concept encourages the development of a sustainable nonchemical method to effectively manage S. zeamais. As an environment-friendly and convenient method, heat treatment has been widely evaluated and applied to controlling insect pests in empty grain storages and processing facilities [12][13][14][15][16]. Usually, the target facility is gradually heated from ambient temperature to 50-60°C during heat treatment [13,[17][18][19][20], which naturally makes the stored product insects experience acclimation to sublethal high temperature. Many studies have shown that acclimation can significantly affect the thermal tolerance of insects [21][22][23].
In addition, most studies have focused on the effects of constant elevated high temperatures on mortality of stored grain insects [18,19]. In comparison, the effects of acclimation to sublethal temperatures on the mortality of stored-product insects are poorly understood, and such information is important for developing effective heat treatment protocols, and understanding responses to thermal stress and the adaptive evolution in response to climate warming [24,25]. In current study, the hypothesis is that acclimation to sublethal temperatures can enhance the heat tolerance of S. zeamais adults and decrease their mortality. And the objective of this study is to evaluate the effect of acclimation to sublethal temperature on subsequent susceptibility of S. zeamais adults to lethal high temperatures.

Insects
S. zeamais was cultured in a controlled temperature and humidity chamber (27±2°C, 75±5% relative humidity and 12:12 L:D) without exposure to any insecticide at the Institute of Stored Product Insects of Henan University of Technology, Zhengzhou, China. The food media used were washed, sterilized whole wheat grains at about 13.5% equilibrium moisture content. The cultivar of the wheat used as a food media was Zhoumai 22. Healthy and 1-2-week old adults were randomly chosen for bioassays.
Insects are ectotherms, and ambient temperature therefore significantly affects their life activities. The optimal temperatures are 25-33°C for growth and reproduction of most stored product insects. Our preliminary experiment results showed that the S. zeamais adults could withstand a long-term heat stress before they eventually died at 36°C. Ma and Ma [21] acclimated two aphid species, Sitobion avenae and Rhopalosiphum padi at 36°C for testing their heat-escape temperatures. Meanwhile, heat treatment usually raises the ambient temperature of the target facility to 50-60°C, and the high exposure temperatures of 43, 47, 51, and 55°C frequently occur in the heating process. Thus, we selected 36°C as a short-term sublethal acclimation temperature, and 43, 47, 51, and 55°C as high exposure temperatures.

Statistical analysis
S. zeamais adult mortality data after exposure to different high temperatures for varying periods were calculated as percentages. Mean ± SE mortality of the control adults in each combination of exposure temperature and exposure time was 0.0 ± 0.0. Therefore, treatment mortality data were not corrected for control mortality [26]. Treatment percentage mortalities were transformed to arcsine square-root values for two-way analysis of variance (ANOVA) procedure with insect mortality as response variable and acclimation time, and exposure time as fixed effects, and the mean mortalities were compared and separated by Scheffe's test at p = 0.05. These analyses were performed using SPSS Version 16.0 software.

Results
The mortality of S. zeamais at 43°C The mortality of S. zeamais adults significantly increased with increasing exposure time at 43°C (Table 1). Compared with the mortality of S. zeamais adults without acclimation (set as a control, 0 h), the mortality of S. zeamais adults with acclimation was significantly lower, especially when the exposure times were more than 365 min at 43°C. The acclimation time, exposure time, and the interaction between the acclimation time and exposure time significantly affected the mortality of S. zeamais adults at p<0.05 level ( Table 2).
The mortality of S. zeamais at 47°C The mortality of S. zeamais adults exposed to 47°C is listed in Table 3. The mortality of S. zeamais adults also significantly increased with increasing exposure time at 47°C. The mortality of S. zeamais with acclimation was significantly lower than that of S. zeamais without acclimation (control) to sublethal high temperature, especially when the exposure times were more than 40 min at 47°C. The acclimation time, exposure time, and the interaction between the acclimation time and exposure time significantly affected the mortality of S. zeamais adults at p<0.05 level ( Table 4).
The mortality of S. zeamais at 51°C Table 5 shows the mortality of S. zeamais adults exposed to 51°C. The mortality of S. zeamais also significantly increased with increasing exposure time at 51°C. The mortality of S. zeamais with acclimation was significantly lower than that of S. zeamais without acclimation (control) to sublethal high temperature, especially when the exposure times were 2, 5, and 5.5 min at 51°C. The acclimation time and exposure time significantly affected the mortality of S. zeamais adults at p<0.05 level, and the interaction between the acclimation time and exposure time had no significant effect on the mortality of S. zeamais adults ( Table 6).
The mortality of S. zeamais at 55°C Table 7 shows the mortality of S. zeamais adults exposed to 55°C. The mortality of S. zeamais also significantly increased with increasing exposure time at 55°C. The mortality of S. zeamais Table 2. Two way analysis of variance (ANOVA) parameters for main effects and associated interactions for the mortality of S. zeamais exposed to 43°C. with acclimation was significantly lower than that of S. zeamais without acclimation (control) to sublethal high temperature, especially when the exposure times were 50, 60, 70, 80, 90, and 110 s at 55°C. The acclimation time and exposure time significantly affected the mortality of S. zeamais adults at p<0.05 level, and the interaction between the acclimation time and exposure time had no significant effect on the mortality of S. zeamais adults (Table 8).

Discussion
The current study indicated that acclimation to sublethal high temperature could significantly enhance the survival of S. zeamais adults subsequently exposed to lethal high temperatures and reduce their mortality. In other words, acclimation to sublethal high temperature significantly enhanced the heat tolerance level of S. zeamais adults and reduced their subsequent susceptibility to lethal high temperatures.  Table 6. Two way analysis of variance (ANOVA) parameters for main effects and associated interactions for the mortality of S. zeamais exposed to 51°C. The susceptibility of S. zeamais to lethal high temperatures was affected by various treatment factors, including insect strain, developmental stage, temperature-time combination, acclimation to sublethal high temperature, heating rate and treatment condition [27]. Li et al. [27] reported that the slowest heating rate (0.1°C/min) achieved the highest mortality of S. zeamais in controlled atmosphere conditions but lowest mortality in regular air conditions. In the present study, we investigated the effect of acclimation to sublethal temperature on subsequent susceptibility of S. zeamais adults to lethal high temperatures. The effect of acclimation on different strains and developmental stages (egg, larva, and pupa stages) of S. zeamais needs to be further investigated in the future.
The ability of insects to deal with heat stress can be achieved through physiological and biochemical mechanisms [28][29][30][31], including short-term processes such as acclimation or longterm processes such as evolutionary adaptation [32] (Huey, 2010). Short-term heat acclimation in laboratory shapes part of the insect responses to their ambient environment, which may involve physiological and biochemical changes to cope with environmental temperature variation [29,33,34]. Tungjitwitayakul et al. [35] reported that heat shock at 30-50°C for 1 h increased three heat shock protein (hsp) genes expression in S. zeamais as follows: Szhsp70 > Szhsp90 > Szhsc70. In the present study, S. zeamais adults were acclimated at 36°C for 0 (control), 1, 3, and 5 h. Probably, the three hsps and other hsps, as well as some other metabolic regulation pathways, were responsible for the enhanced heat tolerance of S. zeamais adults with acclimation. Furthermore, when S. zeamais adults were exposed to 43, 47, 51, or 55°C for various time intervals, no significant differences were observed among the mortalities of S. zeamais adults with 1, 3, and 5 h of acclimation to 36°C, indicating that similar physiological and biochemical mechanisms were involved in the increased thermal tolerance of S. zeamais adults with acclimation. Therefore, the physiological and biochemical adaptation mechanisms of S. zeamais adults with acclimation necessarily deserves to be further investigated, which are in favor of understanding responses to thermal stress and the adaptation evolution in response to ongoing climate warming [25].
Generally, the temperatures are not evenly distributed in the whole treated facility during heat treatment. This inevitably results in the acclimation in stored product insects to sublethal temperature during heat treatment process, which will enhance their survival in the heat stress environment. The survived individuals will form a threat to the stored products after the heat treatment. According to the present research results, the whole target facility should be elevated to over 50°C as soon as possible to avoid reducing the disinfestation effectiveness resulting from the acclimation of stored product insects to sublethal temperature. Table 8. Two way analysis of variance (ANOVA) parameters for main effects and associated interactions for the mortality of S. zeamais exposed to 55°C.