Effects of 50 ° C temperature on Sitophilus granarius ( L . ) , Sitophilus oryzae ( L . ) and Sitophilus zeamais ( Motsch . )

Adults of laboratory populations of granary weevil (S. granarius), rice weevil (S. oryzae) and maize weevil (S. zeamais) were exposed at the temperature of 50°C to determine lethal time (parameters LT20, LT50 and LT99) and progeny production/reduction in F1 generation at mentioned temperature. For each exposure and each species 25 adults aged 2-5 weeks, in four replicates, were used. In the first treatment, the insects were exposed for 6, 10, 14, 16, 18, 20, 22, 26 and 30 min in plastic dishes (V=200 cm3) with 1.8 ± 0.2 g of untreated coarse wheat. In the second treatment, in dishes with 100 g of untreated wheat grain the adults were exposed for 90, 100, 110, 120, 130, 140, 150, 165 and 180 min. After the exposure, the adults were placed in 100 g of untreated wheat grain, in four replicates, at 25 ± 1oC and 60 ± 5% r.h., for recovery. After one, two and seven days of recovery, the weevils were separated by sifting of wheat, and the mortality was determined, and after total period of eight weeks from the exposure of parents, the effect on progeny in F1 generation was determined. Lethal time for weevils was determined by probit analysis, and progeny production/reduction by analysis of variance. After weevils exposure in coarse wheat and after one, two and seven days of recovery, S. oryzae adults were the most susceptible (LT50 12.48-13.68 min), and the most tolerant were adults of S. granarius (LT50 17.79-20.89 min). After insects exposure in wheat grain, the most susceptible were S. granarius and S. oryzae (LT50 107.11-120.73 min), and the most tolerant S. zeamais (LT50 139.90-155.35 min). After exposure of parents of all three weevil species, in coarse wheat, progeny reduction at 100% level is after 22 min. However, after exposure of parents of all three weevil species in wheat grain, progeny reduction at 100% level in S. granarius is after 130 min, in S. oryzae after 150 min, and at 99.7% level in S. zeamais after 180 min. The investigations indicated that short-term exposure of weevils from Sitophilus genus at the temperature of 50°C adversely affects their survival and progeny production, as well as that there is a potential for its successful use as a physical measure in control of storage pest insects.

Having in mind that insects are poikilotherm organisms, and that their development and survival greatly depend on environmental temperature, high temperatures (as a physical measure) alone, or in combination with some other control measures against storage pests are being used in the last few years (Mahroof et al., 2003a(Mahroof et al., , 2003b;;Roesli et al., 2003;Kljajić and Andrić, 2010).Temperature of 50°C is considered to be the most effective (Wright et al., 2002;Mahroof et al., 2003aMahroof et al., , 2003b;;Roesli et al., 2003), because relatively fast, without use of chemicals and risk of resistance development a total disinfestation is obtained (Beckett et al., 2007).There is a lot of literature data on the effects of high temperatures: on survival of storage insects (Kirkpatrick and Tilton, 1972;Fields, 1992;Kljajić et al., 1996;Beckett et al., 1998), on development of insects (Fields, 1992;Beckett et al., 1998;Wright et al., 2002;Mahroof et al., 2003b;Loganathan et al., 2011), on toxicity of contact insecticides in combined application (Kljajić et al., 2009), as well as on physiological, biochemical, and other consequences that occur when storage insects are exposed to high temperatures (Fields, 1992;Neven, 2000).
The objective of this paper was to investigate and compare the effects of 50°C temperature on S. granarius, S. oryzae and S. zeamais under laboratory conditions, in coarse wheat (1.8 g) and wheat grain (100 g), by determination of lethal parameters (LT 20 , LT 50 and LT 99 ), as well as effects on progeny production/reduction in F 1 generation.

Test insects
Adults of laboratory populations of S. granarius, S. oryzae and S. zeamais reared in insectarium, according to methods described by Harein and Soderstrom (1966) and Davis and Bry (1985), were used in the study.Weevils, S. granarius and S. oryzae were reared on soft whole grain wheat, and S. zeamais on the whole grain maize, moisture of 12%, in glass jars (volume 2.5 dm 3 ), at 25 ± 1°C and 60 ± 5% r.h.
Adult insects aged two to five weeks in the unknown sex ratio, were used in the experiment.

Bioassay
Effects of 50°C temperature were investigated in two treatments according to adjusted method used by Mahroof et al. (2003b).
First treatment -in plastic dishes (V=200 cm 3 ) 1.8 ± 0.2 g of untreated coarse wheat grains was added, to avoid direct contact of insects with the dish and too rapid reaction of insects to the heat, i.e. conditions of emty storages were imitated.Exposure intervals of adult test insects were 6, 10, 14, 16, 18, 20, 22, 26 and 30 min.Second treatment -in plastic dishes (V=200 cm 3 ) 100 g of untreated wheat grain was added with an average temperature of 25 ± 1°C and moisture content of 11.5 ± 0.5%.Exposure intervals of adult test insects were 90, 100, 110, 120, 130, 140, 150, 165 and 180 min.Both investigated treatments were set up in four replicates with 25 insects, for each exposure interval, after which they were put in the incubator (MRC, Israel) at 50 ± 1°C and 11 ± 1% r.h.
After each exposure, the weevils were put in dishes with 100 g of untreated wheat grains, under conditions of 25 ± 1ºC and 60 ± 5% r.h. for recovery.Weevil mortality was assessed after one, two and seven days of recovery.On the seventh day, all the weevils (alive and dead) were removed by sifting, to determine the effects on progeny production/reduction in F 1 generation after eight weeks of exposure of parents.The same procedure was applied to control weevils except that they were not exposed at 50°C temperature.

Data analysis
The mortality data were corrected for those in controls using the Abbott (1925) formula and were processed by probit analysis according to method described by Finney (1971) using computer software developed by Raymond (1985).Before analysis progeny number in F 1 generation was transformed using log (x + 1) and then were statistically compared using one-way ANOVA and the significance of mean differences between treatments and control was determined by Fisher's LSD test at P < 0.05 (Sokal and Rohlf, 1995).However, untransformed means and standard errors are shown in the tables.The reduction in progeny size against the control is shown as a percentation (PR %) according to formula used in similar studies by Tapondjou et al. (2002).

RESULTS
The results obtained for lethality (LT 20 , LT 50 and LT 99 ) to weevils after different recovery periods from their exposure at 50ºC in coarse wheat are shown in Table 1, and in wheat grain in Table 2.According to nominal values of lethal parameters at levels LT 20 , LT 50 and LT 99, realized after exposure in dishes with coarse wheat, regardless recovery period, it can be concluded that S. oryzae adults are the most susceptible (LT 50 12.48-13.68min), and the most tolerant are adults of S. granarius (LT 50 17.79-20.89min).However, given the confidence intervals for obtained LT values, there are no statistically significant differences among adults of all three weevil species at 50ºC.Also, after insects exposure to wheat grain, according to nominal values at LT 20 , LT 50 and LT 99 levels, it can be concluded that S. granarius (LT 50 114.32-119.87min) and S. oryzae (LT 50 107.11-120.73min) are the most susceptible, and the most tolerant (LT 50 139.90-155.35min) is S. zeamais, but the differences, regardless recovery period, are not statistically significant.
After different exposures of parent weevils at 50°C, significant progeny reduction in both tested treatments was recorded, and the results obtained i.e. average number of offsprings and calculated values of progeny reduction (PR %) are shown in Tables 3 and 4.
According to the results shown in Table 3, after 10 min exposure nominally the lowest number of offsprings in all three species of weevil parents in coarse wheat was in S. granarius and S. oryzae (130.0;131.5;PR=53.9 and 44.7%), and the highest in S. zeamais (156.5;PR=30.5%),but the differences between them were not significant.After exposure of 20 min, progeny reduction was 98.8-99.3% in all three weevil species, and after 22 min of exposure 100%.After 10 min exposure of S. granarius parents in coarse wheat at 50°C, the average number of offsprings (130.0) was 2.2-fold lower compared to average number of offsprings in control (282.3), and after 20 min even 80.6-fold lower.In S. oryzae after 10 min of parents exposure the average number of offsprings was 1.8-fold lower compared to control (238.0), and after 20 min even 132.2-foldlower.In S. zeamais after 10 min exposure the average number of offsprings was 1.4-fold lower compared to control (225.3), and after 20 min 90-fold lower.
After 90 min exposure of all three species of weevil parents in wheat grain (Table 4), nominally the lowest number of offsprings was recorded in S. granarius (90.5;PR=67.9%), about 48% more than in S. oryzae (133.5;PR=43.9%), and the highest (80% more) in S. zeamais (159.3;PR=29.3%),yet with no significant differences.After 180 min exposure, progeny reduction was high in all three weevil species, 99.7% in S. zeamais, and 100% in S. granarius and S. oryzae.In S. zeamais a certain variability in progeny production can be observed, which is not in direct connection with exposure intervals of weevil parents.

DISCUSSION
In general, the results obtained in this experiment are in agreement with results of recent studies on the efficacy of high temperatures in storage insects control, as well as with the claim that temperature of 50°C is minimum effective temperature for direct use in protection of stored products, particularly in empty storage facilities.Although it has to be pointed out that this is a very costly measure with a high risk of damaging equipment and sensitive materials at longer heating intervals (Fields and White, 2002;Beckett and Morton, 2003;Mahroof et al., 2005;Arthur, 2006;Tilley et al., 2007b).
In previous studies (Kirkpatrick and Tilton, 1972) it was found that the most tolerant to high temperatures is S. zeamais, less S. oryzae, and the least S. granarius, which is in agreement with the results obtained in our experiment.Kljajić et al. (1996) found that after 24 h recovery from exposure of S. granarius adults, in empty glass dishes covered with Petri dishes, at high temperatures (45, 50, 55 and 60ºC), LT 50 is 31 min at 50ºC, and LT 95 46 min.This is about 1.6-fold slower action than in our experiment where S. granarius was exposed in coarse wheat, and about 3.7-fold faster than when granary weevils were exposed in 100 g of wheat grain.Besides use of plastic dishes without cover in this investigation, the reason for expressed differences could be a higher relative air humidity (20 ± 2%) in the incubator.Beckett et al. (1998) by exposing S. oryzae adults at 48°C in wheat grain found that for 99% mortality it takes 38 min, which is about 5.1-fold faster than in our investigation where the weevils were exposed in 100 g of wheat grain.The same authors found that the second larval stage of rice weevil is the most tolerant to high temperatures (LT 50 = 144 min).
Findings on effects of high temperatures on other species of storage insects are different.For example, Fields (1992) and Fields and Muir (1996) found that for survival of 2% of Tribolium castaneum (Herbst) adults and 3% of Rhyzopertha dominica (F.) adults at temperature of 57ºC it takes 32 seconds.Mahroof et al. (2003b) investigated effect of high temperatures on different development stages of T. castaneum and found that young larvae were the most tolerant to temperatures above 50ºC and that for 99% mortality it took even 7.2 h.The same authors recorded a negative impact of 50ºC temperature on progeny production of T. castaneum.From the studies conducted by Arthur (2006) and Tilley et al. (2007a) it can be concluded that exposures shorter than 2 h at 51°C and 50°C cause a 100% mortality of all development stages of T. castaneum and Tribolium confusum (DuVal) and adults of T. castaneum, S. oryzae and R. dominica.
In broader context of consideration of advantages and disadvantages of 50°C temperature use, the optimization of costs and risks of equipment and sensitive materials damaging is also very important, and therefore we point to some research results on direct effects of short-term exposure of storage insects to high temperatures, where populations of different densities and with altered susceptibility to insecticides were also included.From the literature data summarized by Fields (1992) it can be seen that 30% of S. granarius adults recover after exposure of 20 s at 49°C, and 10% after 5 s exposure at 52°C, while 62% of S. oryzae specimens recover after 108 seconds exposure at 50°C, and 33% after 30 s exposure at 52°C.Kljajić et al. (1996) after the exposure of laboratory population of S. granarius, from population densities of 500, 1000 and 2000 adults/1500 g grains, found that LT 50 is 19-26 min at 50°C after recovery of 24 h, and LT 95 22-37 min, which is in agreement with our experimental results if confidence intervals are cosidered (Table 1).Kljajić et al. (2009) investigated the effect of 50ºC temperature on insecticide toxicity to S. granarius adults (one laboratory and two field populations resistant to deltamethrin and pirimifos-methyl) and found that because of altered susceptibility to pirimifos-methyl for total paralysis of the field weevils it took 22% more time than for laboratory population.This reaction was explained by slower physiological-biochemical processes in speciments with altered susceptibility to insecticides, and that the dynamics is not significantly affected by prior direct short-term exposure at 50ºC.
Besides determined LT parameters, the results that show that 50°C temperature greatly affects weevils progeny production and that delayed effect on stored wheat can be obtained by its use are very important in this paper.Due to differences in the concept, objectives and methodologies used, it is difficult to adequately compare the results obtained in our experiment with the relevant data available in literature.However, generally it can be concluded that for all three species of Sitophilus genus the use of 50°C temperature is more effective when speciments are outside wheat grains, for example in an empty storage, than in substrate where the exposure of weevil parents has to be prolonged, in S. granarius for 110 min, in S. oryzae for 130 min and in S. zeamais for over 160 min, to achieve total progeny reduction.
With an aim to find better solutions in control of mentioned and other storage insects, in the future studies it is necessary to investigate the use of 50°C temperature in different substrate masses on several storage pests (primary and secondary) comparatively.To reduce the cost of 50°C temperature treatments and the risk of equipment and sensitive materials damaging, it is necessary to include use of temperature in combination with, for example insecticides of natural origin, especially with inert dusts as one more effective physical measure for pest insects management in stored products.

Table 1 .
Lethal time (LT) for S. granarius, S. oryzae and S. zeamais adults after exposure at 50°C in coarse wheat

Table 2 .
Lethal time (LT) for S. granarius, S. oryzae and S. zeamais adults after exposure at 50°C in wheat grain

Table 3 .
Average number of offsprings (No.±SE) and progeny reduction (PR, %) S. granarius, S. oryzae and S. zeamais in F 1 generation after exposure of parents in coarse wheat at 50°C * For each species, means within columns followed by the same letter are not significantly different (p<0.05)

Table 4 .
Average number of offsprings (No.±SE) and progeny reduction (PR, %) S. granarius, S. oryzae and S. zeamais in F 1 generation after exposure of parents in wheat grain at 50°C