Effects of ultraviolet-C and microwave irradiation on the expression of heat shock protein genes in the maize weevil ( Coleoptera : Curculionidae )

The maize weevil, Sitophilus zeamais (Motschulsky), is a major pest of stored grain kernels. Irradiation is an established technique for controlling insects in stored grain and is a major stress factor affecting these insects. Since heat shock protein (hsp) genes respond to this stress, we proposed that hsps may be associated with irradiated stress tolerance in S. zeamais. The responses of the maize weevil to exposure to ultraviolet-C (UV-C) and microwave irradiation were assessed at four developmental stages: egg, larva, pupa and adult. The results revealed that exposure to UV-C (254 nm, < 8 h) did not affect the survival of the maize weevils; however, Szhsp70, Szhsc70 and Szhsp90 mRNA levels signifi cantly increased during the fi rst 1 h of UV-C exposure. The median lethal time (LT50) of exposure to microwave irradiation indicated that the adult stage was more tolerant of microwave irradiation than the other developmental stages. Microwave irradiation enhanced the expression of the three hsps, but the intensity of up-regulation differed among the three genes, with Szhsp70 the most highly up-regulated. Our experiments revealed that UV-C and microwave irradiation infl uenced the expression profi le of hsp genes in S. zeamais. At the tissue level, the gene responses to UV-C and microwave irradiation varied greatly in different tissues.


INTRODUCTION
The maize weevil, Sitophilus zeamais (Motschulsky), is one of the most important insect pests of maize and other cereals during storage (Noomhorm et al., 2009;Vadivambal et al., 2010).The immature stages of this weevil develop inside the kernels, which results in a loss of weight and deterioration of the grain (Storey, 1987;Noomhorm et al., 2009).Methyl bromide and phosphine are commonly applied worldwide as effective methods to control infestations of storage pests.However, these have resulted in serious environmental damage and are a hazard to human health.Moreover, these pests are becoming increasingly more resistant to phosphine fumigation (Zhao et al., 2007).
Ultraviolet (UV) irradiation has been widely used to kill insects at various stages in their life cycles (Baden et al., 1996).The shortest wavelength radiation, UV-C (100-280 nm), is the most active and has the greatest potential to induce biological damage (Pattison & Davies, 2006).The effi cacy of UV-C against some beetles and mite pests in stored products is well established (Calderon & Navarro, 1971;Furaki et al., 2007;Lah et al., 2012), with sensitivities depending on the species and dose.This has led to a number of investigators considering the possibility of larvae, pupae or adults were placed in 7 × 10 cm plastic zip top bags (Lu et al., 2010(Lu et al., , 2011)).The bags containing the different insect specimens were treated with microwave irradiation at 0, 300, 450, 600, 700 or 800 W for 5-to 100-s.After each treatment, the weevils were removed from the bags and kept for examination.Adults were kept at the standard rearing conditions, and mortality was recorded 1 d following the treatment.The treated insects were transferred onto a sheet of paper and rubbed gently with a paintbrush number 0. Adults were declared dead if they did not respond to the rubbing.Eggs, larvae and pupae which were inside the rice grains were kept for 45, 25 and 15 d following the treatment, respectively, and the number of adults that emerged was record, from which the mortality rate was calculated.

Time-dependent expression of Szhsps
Because UV-C irradiation did not affect the survival of maize weevil adults, we determined its effect on the expression of Szhsps.Rice grains were opened under a stereo microscope to remove the eggs, larvae and pupae, which were placed in zip top bags.Each stage was irradiated for 0, 15, 30, 45 and 60 min.Since the LT 50 after microwave irradiation differed for the four developmental stages, rice grains containing eggs, larvae, pupae and adults (n = 20) were placed in separate zip top bags and exposed to microwave power at 300 W for 5, 10, 15, 20 and 25 s for eggs, at 10, 15, 20, 25 and 30 s for larvae and pupae, and at 20, 40, 60, 80 and 100 s for adults.For each stage this was repeated three times.Whole bodies of insect specimens were subjected to RNA extraction immediately following UV-C and microwave irradiation.

RNA isolation and cDNA synthesis
Immediately after treatment, total RNA was isolated from whole bodies of insect specimens or adult tissues using Ribo-Zol TM RNA extraction reagent (Amresco, OH, USA) following the supplier's instructions.Total RNA was treated with RNasefree DNaseΙ (Thermo Scientifi c, MA, USA) and then subjected to reverse transcription (RT) using oligodT and M-MuLV reverse transcriptase (Sibenzyme, Novosibirsk, Russia).

Tissue distribution of Szhsps
Adults were placed in zip top bags and irradiated under UV-C for 60 min or microwave power at 300 W for 60 s.Immediately after treatment, 30 adults were dissected in Ringer's solution (128 mM NaCl, 18 mM CaCl 2 , 1.3 mM KCl and 2.3 mM NaHCO 3 ) and their ovaries, testes, gut, fat bodies, subesophageal ganglion and Malpighian tubules removed, which were then immediately individually homogenized in RiboZol TM RNA extraction reagent (Amresco, OH, USA) to obtain the total RNA.
UV and microwave irradiation is generally considered to be a common and signifi cant stress affecting animals (Sang et al., 2012).The rice weevil, S. oryzae, is one of the most susceptible species to radiation (Cornwell et al., 1957).Since the maize weevil, S. zeamais, belongs to the same genus, it is assumed that similar hsps are involved in the molecular response of S. zeamais to UV-C and microwave irradiation.Numerous studies report that irradiation induces the expression of the hsp70 gene in mammalian cells (Calini et al., 2003), hsp70 in chick embryos (Shallom et al., 2002) and synthesis of hsp25 proteins in mouse cells (Lee et al., 2006).There are, however, little data on the effects of irradiation on insect hsp genes.
In this study, we recorded the mortality of S. zeamais at four developmental stages, egg, larva, pupa and adult, after exposure to UV-C and microwave irradiation.We also determined the response of maize weevil to UV-C and microwave irradiation in terms of changes in expression of three Szhsp genes, hsp70, hsc70 and hsp90, at individual and tissue levels.

Test insects
Stock cultures of S. zeamais were established and maintained in the laboratory for 1 yr in the dark at 28 ± 1°C and a 65 ± 5% relative humidity.Weevils were reared on heat-sterilized brown rice grains in a wide-mouthed glass jar covered with muslin cloth held in position by rubber bands.To ensure their proper development, insects were kept under moderately crowded conditions.The life cycle of S. zeamais at 28°C was as follows: egg stage, 3 days; larval stage in which the fourth instar is the last larval instar, approximately 22 days; pupal stage, 7 days; adult stage, approximately 6 months.Three day old eggs, 21 day old larvae, 3 day old pupae and 10 day old adults were used in all experiments.The 21 day old larvae became prepupae on day 22 and pupated the following day.

UV-C irradiation
The irradiation source was a 17-watt UV germicidal lamp (TUV F17T8, Philips, Amsterdam, The Netherlands) measuring 58 × 2.5 cm, emitting irradiation at a wavelength of 254 nm.The lamp was fi xed to the ceiling of a test chamber (90 × 60 × 55 cm).The stage on which the insects were exposed to irradiation was 5 cm from the surface of the UV lamp.Three replicates of 20 eggs, larvae, pupae or adults were placed in 7 × 10 cm plastic zip top bags.The bags containing insects at the four different stages were each exposed to UV-C irradiation for 0, 15, 30, 45 or 60 min.Bioassays were conducted at 28 ± 1°C.

Microwave treatment
For the microwave treatment, insect specimens were irradiated with microwave energy using a ME711K microwave oven (Samsung, Suwon, S. Korea).The effective length, width and height of the oven were 34.3, 48.9 and 27.5 cm, respectively.The power output of the generator was adjustable, from 0 to 800 W. To determine the median lethal time (LT 50 ) for the microwave treatments, 3 replicates of 20 rice grains containing either eggs, bank: AY131129.1)served as a reference gene.The primers for SzEF-1 were 5ʹ-AATCTCAGGATGGCATGGAG-3ʹ and 5ʹ-CTGGAGTGGAAGACGGAGAG-3ʹ, with a 178 bp product.PCR reactions were carried out on a CFX96 TM real-time system (Bio-Rad, CA, USA) according to the operation manual.The PCR parameters for the melting curve were as follows: 2 min at 95°C; 40 cycles of 5 s at 95°C, 10 s at 60°C and 15 s at 72°C; then 10 s at 95°C, 10 s at 70°C, and 20 s at 96°C (gradient rising to 96°C, with an interval of 0.2°C).The analysis of the qPCR results was performed using the 2 -∆∆Ct method (Livak & Schmittgen, 2001).All samples were normalized with respect to the reference gene to obtain the ∆Ct value (Ct target gene -Ct EF-1 ) and then to the calibrator value (untreated control) to obtain the ∆∆Ct value (∆Ct target -∆Ct calibrator ).The fi nal relative expression was calculated using the formula F = 2 -∆∆Ct (Chen et al., 2014).
The responses of Szhsp70, Szhsp90 and Szhsc70 to UV-C or microwave irradiation at different developmental stages were analyzed by normalizing the amount of each Szhsp mRNA to SzEF-1, after which the normalized value was divided by the normalized value of Szhsp in the control for each developmental stage; the expression of the control was thus set to 1. Expression of the three Szhsps in adult tissues after UV-C (for 60 min) or microwave irradiation (for 60 s) was also analyzed by normalizing the amount of each Szhsp mRNA to that of SzEF-1 in each tissue, after which the normalized value of each Szhsp was then divided by the normalized value of Szhsp in the control.Thus, the normalized expression level of the control was set to 1 in individual tissues.

Data analysis
Mortality data were processed using Probit analysis and SPSS program version 11.5.The data obtained from the different time points were determined using a one-way analysis of variance (ANOVA; SPSS program version 11.5), followed by a LSD multiple range test.Signifi cant differences in tissues between the control (no stress) and treatment groups (UV-C or microwave irradiation) were identifi ed by an independent-sample t test (SPSS program version 11.5).The signifi cance level was set at 0.05 (P < 0.05).

Effects of UV-C and microwave irradiation on mortality
The effect of UV-C irradiation on S. zeamais was fi rst examined in the adults.All adults survived exposure to UV-C irradiation for 1 to 8 h (data not shown), indicating that UV-C had no effect on the survival of S. zeamais.On the other hand, microwave irradiation had a lethal effect on S. zeamais.The LT 50 of S. zeamais after exposure to a series of different microwave powers over various exposure times is shown in Table 1.The LT 50 values at each power differed for the four developmental stages.The results indicate that adults were the most tolerant of microwave irradiation, followed by pupae, larvae and then eggs.The mortality of irradiated weevils was directly proportional to the exposure times.

Effects of UV-C exposure time on Szhsps expression
The time course of Szhsp expression after UV-C irradiation for the four developmental stages is shown in Fig. 1.A time-dependent expression pattern was clearly observed for Szhsp70 in the eggs, larvae and adults, but not in the pupae.After UV-C exposure, the expression of Szhsp70 in the eggs, larvae and adults increased dramatically and peaked at 60 min.Szhsp70 in the pupae increased for 45  min and then decreased for 60 min.Compared with the other three stages, the response of this gene in the pupae was limited.These results indicate that the intensity of the exposure-time response of Szhsp70 varies depending on the developmental stage.The greatest increase was recorded in adults after UV-C irradiation for 60 min, which was 430-fold higher than that recorded in the control (Fig. 1a), followed by 54-, 30-and 8-fold increases in the larvae, eggs and pupae, respectively.UV-C irradiation enhanced Szhsp90 expression in the larvae, pupae and adults, but not in eggs.It was noteworthy that the intensity of the up-regulation was much lower than that of Szhsp70.The increased level of Szhsp90 expression in adults was highest, reaching a 14-fold increase at 60 min (Fig. 1b), followed by 5-and 1.9-fold increases in pupae and larvae, respectively.
Szhsc70 showed a different pattern of expression from that of Szhsp70 and Szhsp90.Szhsc70 expression in eggs was not altered by UV-C irradiation but was downregulated in the larvae and pupae after UV-C irradiation for 15 to 60 min (Fig. 1c).The Szhsc70 expression pattern in the adults differed from that in the eggs, larvae and pupae.The gene expression in irradiated adults increased at 15 min, decreased slightly at 30 min, and exhibited signifi cant upregulation at 45 and 60 min.Szhsc70 showed the highest expression level (4-fold) at 60 min.

Effect of exposure to microwave irradiation on Szhsps expression
Since the LT 50 values of the four developmental stages after microwave irradiation at 300 W differed, the weevils were irradiated for longer than the LT 50 values of each stage.Szhsp70 expression was signifi cantly increased at 15 s in the eggs and pupae and at 20 s in the larvae and adults.Szhsp70 expression levels in the eggs peaked at 20 s and decreased at 25 s of exposure (Fig. 2a).Microwave irradiation enhanced Szhsp70 expression in the larvae, peaking at 20 s, and was then down-regulated at 30 s (Fig. 2b).In the pupae, Szhsp70 expression was high at 15 s, decreased at 20 and 25 s, and then increased slightly at 30 s (Fig. 2c).The Szhsp70 expression level in the adults was relatively high at 20 to 40 s and peaked at 60 s.After 80 to 100 s of exposure, Szhsp70 expression levels decreased and remained at the basal level (Fig. 2d).The maximal intensities of the gene response varied among the developmental stages: there was a 59-fold increase in adults and 44-, 14-, and 5.8-fold increases in larvae, pupae and eggs, respectively.
Szhsp90 expression in the eggs, larvae and adults exhibited a similar pattern to Szhsp70 expression but showed little response to microwave irradiation.The expression levels in eggs, larvae and adults increased by only 1.5-to 2.8-fold relative to the control (Figs 2a, b and d).In contrast to the other three stages, Szhsp90 expression in the pupae was signifi cantly decreased after microwave treatment (Fig. 2c).
Szhsc70 exhibited the opposite expression pattern to that of Szhsp70 and Szhsp90, as it was down-regulated in the eggs, larvae and pupae after microwave irradiation.How-ever, there was a slightly increased expression in the adults, with a maximum level at 60 s after exposure but with only a 2-fold increase relative to the control.

Tissue-specifi c gene responses to UV-C and microwave irradiation
Szhsps responded to UV-C and microwave irradiation in a tissue-specifi c manner (Figs 3 and 4).After UV-C irradiation for 60 min, Szhsp70 was up-regulated in the ovaries and gut by 2.7-and 15.9-fold relative to the control, respectively (Fig. 3a).UV-C enhanced Szhsp90 expression in the testes, subesophageal ganglion and Malpighian tubules (Fig. 3b).In contrast to Szhsp70 and Szhsp90, UV-C suppressed Szhsc70 expression in the ovaries and gut, while its expression in the testes, subesophageal ganglion and Mal-pighian tubules was not affected.Up-regulation of Szhsc70 by UV-C was recorded only in the fat bodies (Fig. 3c).
The responses of Szhsps to microwave irradiation at 300 W for 60 s also varied among the tissues.Microwave irradiation up-regulated Szhsp70 in the gut, testes, ovaries and fat bodies by 198-, 59-, 6.6-and 2-fold compared with the control, respectively (Fig. 4a).Microwave irradiation increased the expression of Szhsc70 only in the fat bodies by ~1.2-fold compared with the control (Fig. 4c), while expression of Szhsp90 in testes was 1.9-fold higher than in the control (Fig. 4b).Szhsps was markedly down-regulated in other tissues, except for Szhsc70 in the Malpighian tubules, which did not respond to microwave irradiation (Fig. 4c).

Effects of UV-C and microwave irradiation on S. zeamais survival
S. zeamais adults exposed to UV-C for 1 to 8 h showed no lethal effects.Since UV-C has a limited ability to penetrate surfaces, its effects are limited to the body surface (Ghanem & Shamma, 2007).In addition, cuticular thickness and protective pigments in the adult epithelium can withstand UV and are important for limiting penetration into the body (Buck & Callaghan, 1999;McCloud & Berenbaum, 1999).This result is consistent with those for the red fl our beetle, Tribolium castaneum (Herbst) (Sang et al., 2012), and the potato aphid, Macrosiphum euphorbiae (Thomas) (Nguyen et al., 2009), in which UV exposure had no lethal effects.
In contrast to UV-C, microwave irradiation was lethal for the maize weevil.The mechanisms involved in the lethal action of microwave irradiation could be due to the high oscillation frequency of water molecules in the bodies of the insects.Microwave heating is based on the transformation of alternating electromagnetic fi eld energy into thermal energy by affecting the polar molecules of a material, and hence it can kill the insects at lower temperatures (Lu et al., 2010).It was clear that increasing the microwave irradiation exposure time caused a signifi cant increase in the mortality of S. zeamais.This suggests that microwave stress had a greater effect than that of UV-C stress.The LT 50 of S. zeamais after microwave irradiation varied according to the developmental stage.Eggs were the most susceptible, followed by larvae, pupae and adults, in that order.Other studies also show that the egg stage of insects are more susceptible to irradiation than the other stages (Tilton & Brower, 1983;Hasan & Khan, 1998;Zhao et al., 2007;Vadivambal et al., 2010).Embryonic cells divide and grow more rapidly than other cells at later stages and this makes them more susceptible to damage from irradiation (Azizoglu et al., 2011).Exposure of adult S. zeamais to different microwave irradiances revealed a higher LT 50 in adults, indicating that the adult stage was the more tolerant of irradiation than the early life stages.

Expression of three Szhsp genes after UV-C and microwave irradiation
The apparent lack of an effect on the survival of adult S. zeamais does not indicate that UV-C had no deleterious effects on the weevils.UV-C irradiation at 254 nm has a higher penetrative capacity than that of other UVs, and the response of Szhsp to UV-C was unique among the three genes evaluated.The speed of the response was substantially different depending on the developmental stage.Szhsp70 and Szhsp90, but not Szhsc70, were upregulated after UV-C irradiation.Szhsp70 and Szhsp90 in adults showed the highest expression levels after the same exposure time, but the increase in expression of Szhsp70 was much higher than that of Szhsp90.This characteristic response of hsp70 seems to be a common feature, as this is also reported for T. castaneum (Sang et al., 2012), in which hsp70 is the most highly up-regulated.UV-C strongly suppressed Szhsc70 expression in the eggs, larvae and pupae, but signifi cantly induced Szhsc70 expression levels in the adults.However, the increase in Szhsc70 expression in the adults was much lower than that of Szhsp70 and Szhsp90.These results indicated that UV-C may induce Szhsp70 and Szhsp90 expression to protect the weevil from UV-C stress.
Similar to the hsp gene responses to UV-C irradiation, the Szhsp response to microwave stress was not uniform for the three genes examined.The timing of the gene responses and the increases in intensity depended on the developmental stage.Szhsp70 was up-regulated promptly in four developmental stages, while Szhsp90 was up-regulated in the eggs, larvae and adults.In contrast, Szhsc70 was down-regulated in the eggs, larvae and pupae.It is reported that microwave irradiation induces an obvious thermal effect (Lu et al., 2010), hence increased temperature may be involved in the up-regulation of Szhsp70 and Szhsp90 expression levels.This characteristic response of hsp70 seems to be a common feature as it is reported in chick embryos (Shallom et al., 2002) and human neuroblastoma SH-SY5Y cells (Calabrò et al., 2012), in which microwave irradiation is able to induce a thermal effect and activate hsp70.
The temperature used to induce maximal hsp expression provides an important clue for our understanding of the limits of temperature tolerance (Huang & Kang, 2007).In S. zeamais, expression of the Szhsp genes after microwave irradiation increased signifi cantly depending on the exposure time but decreased at LT 50 .Exposure times over the LT 50 resulted in a decline in the survival of all stages, which was consistent with decreased expression of Szhsps.This indicates that the ability of Szhsps to act as molecular chaperones might limit their mitigation of the damage caused by high temperatures at LT 50 and above.
In this study, after UV-C irradiation, three Szhsps showed a tissue-specifi c expression pattern.Szhsp70 was expressed only in the ovaries and gut.On the other hand, after microwave irradiation, Szhsp70 was up-regulated in the ovaries, testes and gut.It was noteworthy that Szhsp70 in the gut showed the highest expression level after UV-C and microwave irradiation, and it is also reported that the activation of Szhsp70 gene expression in gut increases after heat and cold treatment (Tungjitwitayakul et al., 2015).Similar to Apis cerana cerana (Fabricius) after UV irradiation, hsp22.6 is detected at higher levels in the midgut than in other tissues tested (Zhang et al., 2014).After UV-C irradiation, the Szhsp90 expression level was increased in the testes, subesophageal ganglion and Malpighian tubules, but only in the testes after microwave irradiation.In contrast to Szhsp70 and Szhsp90, Szhsc70 was up-regulated only in the fat bodies after UV-C and microwave irradiation, indicating that the three Szhsps were uniquely expressed in the different tissues in response to different stressors.
In conclusion, our study shows the responses of three heat shock protein genes in the S. zeamais to UV-C and microwave irradiation.An increase in Szhsp70 and Szhsp90 expression may in turn protect the maize weevils from irradiation damage in a dose-dependent manner.However, the detailed mechanisms of how Szhsp70 and Szhsp90 are involved in the protection pathway need to be further examined using other techniques such as RNA interference.

Fig. 1 .
Fig. 1.The responses of three Szhsps in different developmental stages of S. zeamais to UV-C irradiation.Bars indicate the means of three independent biological replicates and include the standard deviations (S.D).Different letters above the bars indicate signifi cant differences compared with the other exposure times (P < 0.05).

Fig. 2 .
Fig. 2. Responses of three Szhsps in different developmental stages of S. zeamais to microwave irradiation.Bars indicate the means of three independent biological replicates and include the standard deviations (S.D).Different letters above the bars indicate signifi cant differences compared with the other exposure times (P < 0.05).

Fig. 3 .
Fig. 3. Expression of the three Szhsps in adult tissues after exposure to UV-C irradiation for 60 min.OV -ovary; T -testis; G -gut; FB -fat body; SG -subesophageal ganglion; MT -Malpighian tubule.Asterisks above the bars within each tissue indicate signifi cant differences (P < 0.05) between the treatment and control groups.

Fig. 4 .
Fig. 4. Expression of the three Szhsps in adult tissues after exposure to microwave irradiation for 60 s.OV -ovary; T -testis; G -gut; FB -fat body; SG -subesophageal ganglion; MT -Malpighian tubule.Asterisks above the bars within each tissue indicate signifi cant differences (P < 0.05) between the treatment and control groups.
* Asterisk indicates no insects survived microwave irradiation.