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Journal of Stored Products Research

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Calorimetric evaluation of responses of Sitophilus oryzae and Tribolium confusum to elevated temperatures and controlled atmospheres
C.J. Downes a,, C.W. van Epenhuijsen b, R.E. Lill b, J.E. Downes c, A. Carpenter b,1, D. Brash b
a b c

Industrial Research Limited, P.O. Box 31 310, Lower Hutt 5040, New Zealand New Zealand Institute for Crop and Food Research Limited, Private Bag 11600, Palmerston North 4442, New Zealand Division of ICS, Physics Department, Macquarie University, Sydney, NSW 2109, Australia

a r t i c l e in fo
Article history: Accepted 1 March 2008 Keywords: Sitophilus oryzae Tribolium confusum Controlled atmospheres Heat treatment of stored-product insects Microcalorimetry Thermo-gravimetric analysis

abstract
Metabolic heat rates, determined by microcalorimetry, were used to measure the effect of controlled atmospheres (CAs) and elevated temperatures on the stored-product insects Sitophilus oryzae (rice weevil) and Tribolium confusum (confused our beetle). Results for larval and adult stages in air, and in a range of low O2 and/or high CO2 CAs, at temperatures from 15 to 45 1C, showed the general effectiveness of such atmospheres in lowering the lethal temperatures relative to those in air. Effects on adult S. oryzae at 25 1C were explored in more detail in experiments using the following conditions: exposure to anoxic CAs for extended times; exposure to hypoxic CAs; and simulated hermetic storage. A simple scanning calorimetric method was developed for determining lethal temperatures and a combined thermo-gravimetric and differential thermal-analysis method was used to interpret the thermal events, due to loss of water, occurring at and above these temperatures. & 2008 Elsevier Ltd. All rights reserved.

1. Introduction Insects associated with the storage and processing of grain, if uncontrolled, are a major source of spoilage and consequent economic loss. In the early part of the 20th century, heat sterilisation was a common control method for such insects, especially in the United States of America (Dean, 1911). During the past century these heat treatment methods were supplanted by chemical fumigation. More recently, there has been considerable research on reintroducing heat treatment methods to replace methyl bromide and other hazardous compounds. In a recent study of the application of a heat treatment in a pilot feed mill, Roesli et al. (2003) found that achieving temperatures of 50 1C or more for 2835 h killed all stored-product insects in dishes and should be able to eliminate all exposed stages of stored-product insects in a feed mill. A similar study by Mahroof et al. (2003) reported temperature and relative humidity proles during heat treatment and its efcacy against various life stages of Tribolium castaneum (Herbst). Controlled atmospheres (CAs), high in carbon dioxide and/or low in oxygen, have been advocated for many years for preservation of grain and control of insect infestations. Emekci et al. (2002) reported the effect of CAs with reduced oxygen levels

Corresponding author. Fax: +64 4 931 3142.

1

E-mail address: c.downes@irl.cri.nz (C.J. Downes). Deceased, 7 February 2002.

on the stored-product insect T. castaneum, and they referred to earlier papers on the use of CA technology for preserving stored grain. Moreover, Soderstrom et al. (1992) found that for T. castaneum, elevated temperatures combined with CAs can signicantly reduce treatment time. The present study encompasses a proposal that for selected areas of ourmills, a combination of CAs with heat treatment might provide a more effective control method at lower temperatures than when applied in normal air. This is of particular relevance where the 5060 1C temperatures likely to be required for disinfestation in air are at, or above, the maximum safe operating temperature for electronic control equipment and also of glue used in some sieves. The equipment could be surrounded with a plastic tent to contain the CA and then kept at a lower temperature. Against this background, this report covers the effect of changing environmental conditions on the metabolic heat rate (MHR) of Sitophilus oryzae (L.), the rice weevil, which is often used to test the effectiveness of heat treatments because it tolerates elevated temperatures better than some other stored-product insects (Fields, 1992). Less extensive experiments were carried out on Tribolium confusum du Val, the confused our beetle. The possibility that use of CAs will enable lower temperatures to be effective was tested in the rst set of experiments where MHRs in CAs, relative to those in air, were determined for larval and adult stages of both insect species over the temperature range of 1545 1C. Several variants of the calorimetric method were developed to delineate the responses to CAs and temperature in

0022-474X/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2008.03.002

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greater detail, using mostly adult S. oryzae as the test insect. To aid the interpretation of the effect of hypoxic atmospheres, the MHRs were determined for adult S. oryzae in atmospheres containing 0100% O2. It has been proposed (see, for example, Donahaye et al., 1996, and references therein) that control of insects might be achieved by the use of atmospheres depleted in O2 and enriched in CO2, produced naturally by respiration of the insects and the stored products in well-sealed containers and traditionally referred to as hermetic storage. This process was simulated simply in the calorimeter by using a sealed ampoule, with no replenishment of the atmosphere, and measuring the MHR over an extended time as the insects responded to decreasing oxygen levels and the concomitant rising carbon dioxide levels. Previous experiments with Myzus persicae (Sulzer), green peach aphids, showed that after about 6 h in anoxic atmospheres, the MHR, on returning to air, was less than about 50% of the initial value in air at the start of the experiment, and the insects did not survive (Downes et al., 2003). Experiments were therefore done at 25 1C with adult S. oryzae, using anoxic CAs of 100% N2 and 60% CO2+N2, to see if they followed a similar pattern. As an alternative strategy, the effects of hypoxic CAs were investigated. Raising temperature almost invariably increases the rate of a chemical reaction to a marked extent, and for homogeneous processes the specic rate is approximately doubled or trebled for each 10 1C rise of temperature (Glasstone, 1946). Therefore, it is to be expected that the MHR of insects will increase with increase in temperature until one or more biological process cannot be maintained due to the instability of biomolecules, or because of limitations imposed by physical processes such as diffusion. A more or less rapid decline in MHR would then be expected with further increases in temperature. At some temperature, which may be species-specic, the changes will be irreversible and the insect will die. It was therefore of interest to see if any feature of the calorimetric trace of MHR versus temperature corresponded with reported lethal temperatures. Fields (1992) has collected published data on the survival of adult S. oryzae and T. confusum at extreme temperatures. In practically all cases, lethal temperatures were less than 60 1C, though, as expected, the exact temperature depended on exposure time. Heat treatments of stored products use rapid heating and cooling cycles to minimise damage to the product. Lethal temperatures in this context are likely to be higher than those that the insects can tolerate in the long term. A temperature scanning method was developed to determine lethal temperatures of both adult species in air and in a CA. Finally, a combined thermo-gravimetric and differential thermalanalysis technique was used to examine thermal processes occurring at and above the lethal temperature.

about 50% r.h. with a diet of wheat for S. oryzae and wholemeal our:yeast for T. confusum. Tests conrmed that uninfested wheat grains have an insignicant MHR. The life status of the insects at the end of an experiment was determined by observation after placing them in a 5 mL glass vial tted with a stainless steel gauze insert in the plastic lid. Insects were judged alive if they had obvious movement or could be induced to move at least an appendage on gentle prodding with a ne art brush. Dissection of wheat grains for a visual determination of the life status of S. oryzae larvae was not attempted. The air and CAs consisting of 5% O2+N2, 5% O2+5% CO2+N2, 5% O2+60% CO2+N2, 60% CO2+N2, a range of O2+N2 mixtures and oxygen-free N2, were certied gases in steel cylinders (BOC Gases, NZ).

2.2. General techniques In most experiments, the MHR was measured initially under standard conditions of 25 1C in air, and again during the treatment and, when possible, on returning to standard conditions. The major interest was in the large responses in MHR resulting from substantial changes in conditions so we have, in many cases, relied on the results from a particular sample of insects being representative without the need for taking an average of repeat experiments. The similar results that were obtained in those experiments that were replicated support the use of this procedure. We ignored any effects, expected to be minor, of differences in sex or age among insects. In these experiments, the insects had minimal time to acclimatise to the test conditions so, while the results were relevant to our present aims, they may be less useful in dening limits for the insects natural habitat. The long-term viability of insects that survived the treatments was outside the scope of the present study. All calorimetric measurements of MHRs were made using a Hart DSC model 4207 (current designation for this instrument is CSC 4100, Calorimetry Sciences Corporation, Spanish Fork, UT, USA) with nominal 1 cm3 Hastelloys C removable ampoules. The reproducibility was about 12 mW at near ambient temperatures, increasing to about 5 mW at the extremes of temperature used in the present experiments. Data were collected every 10 s. By convention, exothermic processes were designated as negative heat rate values. Enough insects were used in each measurement to give an initial MHR signal of about 100 mW. As an example, an adult S. oryzae typically had a MHR of about 12 to 20 mW at 25 1C in air. To avoid a noisy thermal signal, mobile insects were conned to the bottom of the ampoule with a disk of ne stainless steel mesh. In scanning experiments, the Tian correction, which removes the time constant of the calorimeter, was applied using the software supplied by the manufacturer. A modied lid on the calorimeter ampoule allowed exchange of the atmosphere in the ampoule in situ with air or the desired CA. Insect metabolism results in changes in the ampoule atmosphere, which in turn would inuence the MHR. To avoid a signicant inuence of this effect, the atmosphere in the ampoule must be replenished. If this were to be done continuously, the incoming gas would need to be carefully thermostated to the temperature of the ampoule. A second point is that evaporation or condensation of water results in large thermal effects. To avoid these problems, the ampoule was automatically ushed periodically, typically for 10 min of every hour for extended runs, by including a solenoid valve in the gas line to the ampoule that was controlled by an electronic timer (Crouzets, MLR1). This ushing caused a major disturbance to the calorimetric signal, but only a negligible change in the temperature of the ampoule. There was then ample time for the signal to settle before the cycle was repeated. Dry gases were used for all the experiments except the

2. Methods and materials 2.1. Insect culture techniques and controlled atmospheres Insects sourced from local our mills and grain silos were cultured in the rearing room at the New Zealand Institute for Crop and Food Research at 27 1C and 70% relative humidity (r.h.). Sitophilus oryzae were reared by allowing hundreds of adults to lay eggs in 150 g of untreated whole wheat for 3 days in 500 mL glass jars with stainless steel mesh in the lids. For trials on the larvae, the wheat grains (each grain usually contains one insect) were used 34 weeks after removing the adults. Emerged adults were used for the trials after 5 weeks. Adult T. confusum were placed in wholemeal:yeast mix (20:1, v/v) and allowed to lay eggs for 10 days. The insects were sieved out and emerging larvae and adults were used in the trials. The insects were transferred to the calorimetry laboratory where they were maintained at 23 1C and

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long-term trials for which they were humidied to about 70% r.h. Previous experiments with M. persicae, in which a mass spectrometer was used to analyse the atmosphere of the calorimeter ampoule, indicated the insects established r.h. at about 50% (Downes et al., 2003). The rate of production of metabolic CO2 in atmospheres containing negligible amounts of CO2 can be determined by measuring the additional CO2 heat rate when a small vial of 0.4 M NaOH is included in the calorimeter ampoule with the insects (Criddle and Hansen, 1999). The CO2 production rate was obtained by dividing the CO2 heat rate by 108.5 kJ mol1, which is the heat of reaction of CO2 with aqueous NaOH. 2.3. Techniques for specic experiments 2.3.1. Effect of controlled atmospheres and temperature The MHR was measured in air at 25 1C before replacing with the CA and again measuring the MHR. The MHR was then determined at 15, 20, 25 and 45 1C before the run was terminated. Each of the isothermal temperatures was maintained for 30 min and the ampoule was ushed as the temperature was changed at 1 1C min1. Air was used as a control treatment, with MHRs determined at the additional temperatures of 30, 35 and 40 1C. 2.3.2. Changes in the composition of the atmosphere in a sealedampoule experiment The changes from the initial air-values in the composition of the atmosphere in a sealed ampoule resulting from the metabolism can be calculated as follows. The energy, in joules, produced over any given time can be obtained by integration of the MHR (power in watts) versus time trace. Using these energy values, the concentration of O2 and CO2 in the atmosphere of the ampoule as a function of time can be calculated from the measured volume of the ampoule and the assumptions that in the production of 455 kJ of heat, 1 mol of O2 is consumed and an equal amount of CO2 is produced. The rst assumption incorporates Thorntons rule, which states that the oxidation of nearly all organic compounds produces 455715 kJ of heat for every mole of O2 consumed (Criddle and Hansen, 1999). The validity of the latter assumptions is discussed below. From the total measured heat the weight of the substrate consumed during an experiment, assuming it to be glycogen, can be calculated from the following equation: 2C6 H10 O5 2n 6nO2 6nCO2 5nH2 O For insects kept without food during the experiment, the calculated weight loss should be equal to the measured value if the glycogen assumption is valid. 2.3.3. Scanning experiments to determine lethal temperatures Unless otherwise noted, insects were scanned over the range of 1560 1C at a nominal scan rate of 1 1C min1. From the start of a scan it took about 10 min before a steady state was reached, so the scans were started at 15 1C in order to provide usable data from 25 1C. The thermal signal in the scanning mode included that from the metabolism together with the heat ow associated with changing the temperature of the insects (heat capacity effects) and, potentially, phase changes including evaporation of water, or other volatiles. In an attempt to remove much of the nonmetabolic contribution, a repeat scan was done on the supposedly now-dead insects and subtracted from the rst scan. 2.3.4. Combined thermo-gravimetric and differential thermalanalysis experiments To help explain the results from the scanning experiments, measurements were made using a Simultaneous Thermal Analy-

ser (PL STA 1500) from Polymer Laboratories (Surrey, UK). This instrument provides thermo-gravimetric (TG) data and differential thermal-analysis (DTA) data simultaneously; that is, it measures weight loss and heat ow to or from a sample as it is scanned across a temperature range. Adult S. oryzae were chilled briey to near 4 1C to aid placement in the alumina crucible (4 mm i.d. 3 mm high).

3. Results 3.1. Effect of controlled atmospheres and temperature In Fig. 1ad, the MHR for each temperature and CA is expressed as a percentage of the initial value in air at 25 1C. The deviation from a 100% value for the MHR at 25 1C in air plotted on these graphs resulted from an exposure of the test insects to an initial interval at 15 1C. For both species at 25 1C, there was a greater spread in the response to the oxygen-containing CAs for larvae than for adults. There was considerable variation in the temperature coefcients of the MHRs in air, the MHR for T. confusum adults at 45 1C being 300% of the value at 25 1C, whereas the corresponding values for T. confusum larvae and S. oryzae adults were about 160%. Nearly all these specimens survived the brief exposure to 45 1C in air. The response of S. oryzae larvae in air was more complicated. Between 15 and 30 1C the MHR temperature coefcient was positive, constant and large. The MHRs at 35 and 40 1C were constant initially, but decreased rapidly in the second half of the observation time. The two plotted values are means of data, each having a spread of about 30 percentage points around the mean. By contrast, the trace at 45 1C showed very little variation. On returning to 25 1C, the MHR (not shown in Fig. 1b) was 38% of the original. Taken together, these results suggest that the larvae were severely stressed as the temperature increased above 30 1C and were probably dead at 45 1C. The MHRs of S. oryzae larvae conned in wheat grains responded to changes in the atmosphere within the times taken for the calorimeter to reach thermal equilibrium, in a similar fashion to free insects. However, on changing the temperature from 25 to 45 1C, in one step, a longer thermal equilibration time was required because of the greater thermal mass due to the presence of the wheat grains. For both adult and larval S. oryzae, the MHRs at 45 1C for the 5% O2+60% CO2+N2 CA were greater than for the other CAs. The MHR plotted in Fig. 1a for the adults was that at the standard equilibration time. However, at this time, the MHR was decreasing rapidly and all insects were dead on removal from the calorimeter at the end of the experiment. In these short-term experiments, only the 5% O2+60% CO2+N2 and 60% CO2+N2 CAs were lethal to the adults at 45 1C. It is suspected, based on the MHR values, but not conrmed by other means, that the larvae did not survive in any of the atmospheres at 45 1C. For both adults and larvae, increasing concentrations of CO2 in 5% O2 gave higher MHRs at 45 1C (expressed as a percentage of the exothermic 25 1C value in air). The negative values shown for the 60% CO2+N2 CA indicate endothermic heat rates (i.e. positive observed heat rates). The results for T. confusum adults in CAs shown in Fig. 1c are closest to what might have been expectedthat with increasing concentrations of CO2 in 5% O2 the absolute MHR decreased. The data for T. confusum larvae, shown in Fig. 1d, followed a similar pattern except that, as for S. oryzae adults and larvae, the presence of 60% CO2 in 5% O2 raised the MHR at 45 1C above the value for 5% CO2. In these short-term exposures the larvae were more resistant than the adults, with only the 60% CO2+N2 CA resulting in the death of the insects. Fig. 1ad shows that replacing air with 5% O2+N2 resulted in a decrease in the MHR at 25 1C for adults and larvae of both species.

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160 140 Metabolic heat rate (%) 120 100 80 60 40 20 0 -20

S. oryzae Adults
air 5% O2 + N2 5% O2 + 5% CO2 + N2 5% O2 + 60% CO2 + N2 60% CO2 + N2

160 8A 7D 6A Metabolic heat rate (%) 140 120 100 80 60 40 20 0 8D -20

S. oryzae Larvae air 5% O2 + N2 5% O2 + 5% CO2 + N2 5% O2 + 60% CO2 + N2 60% CO2 + N2

8A

15

20

25 30 35 Temperature (C)

40

45

15

20

25 30 35 Temperature (C)

40

45

350
T. confusum Adults

180
air 5% O2 + N2 5% O2 + 5% CO2 + N2 5% O2 + 60% CO2 + N2 60% CO2 + N2

300 Metabolic heat rate (%) 250 200 150 100

5A 1D Metabolic heat rate (%)

160 140 120 100 80 60 40 20 0 -20

T. confusum Larvae
air 5% O2 + N2 5% O2 + 5% CO2 + N2 5% O2 + 60% CO2 + N2 60% CO2 + N2

8A

8A 8A

5A 1D

5A 1D

7A 1I

8D 50 0 15 20 25 30 35 Temperature (C) 40 8D 45

8D

15

20

25 30 35 Temperature (C)

40

45

Fig. 1. (ad) Metabolic heat rates (MHR), at 1545 1C, in air and controlled atmospheres, expressed as a percentage of the initial value in air at 25 1C. Run conditions: isothermal at 25 1C in air to give reference MHR, then 30 min at each temperature from 15 to 45 1C, temperature increased between isothermal steps at 1 1C min1. Life status determined at end of run: A alive, D dead, I dead, injured by mesh. (a) Adult Sitophilus oryzae; (b) larval Sitophilus oryzae (45 min at 45 1C, life status not determined (see text)); (c) adult Tribolium confusum; (d) larval Tribolium confusum.

100 Metabolic heat rate (%) 80 60 40 20

Sitophilus oryzae adults were examined in more detail by measuring the MHR over a range of concentrations of O2 in N2 containing minimal amounts of CO2 from the metabolism over the time of the measurements. The results in Fig. 2 show a reduction in the O2 concentration from 21% to 5% resulted in a decrease in MHR of only about 20%, while a decrease in MHR of about 50% occurred at about 2% O2. Tests using 100% O2, with results not recorded here, showed the MHR to be practically identical to the value in air.

3.2. Effect of hypoxic and anoxic atmospheres

0 0 5 10 15 Oxygen concentration (%) 20 25

Fig. 2. Metabolic heat rate for adult Sitophilus oryzae, at 25 1C, expressed as a percentage of the value in air, as a function of oxygen concentration.

3.2.1. Changes in the composition of the atmosphere in a sealedampoule experiment The results for adult S. oryzae in a sealed-ampoule experiment are shown in Fig. 3. The rst 1500 s of the run, when the calorimeter was reaching a steady state, have been omitted from the graphs in Fig. 3. Allowance was made for this when calculating

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0 20 Carbon dioxide concentration -20 -40 Metabolic heat rate 10 -60 -80 -100 5 Oxygen concentration -120 -140 80000 Metabolic heat rate (W)

Concentration (%)

15

0 10000 20000 30000 40000 50000 Time (s) 60000 70000

Fig. 3. Metabolic heat rate for adult Sitophilus oryzae, at 25 1C, and calculated oxygen and carbon dioxide concentrations as a function of time, in an experiment to simulate hermetic storage.

the O2 and CO2 concentrations. Only the data for the rst 80,000 s are shown from a total run time of 307,152 s (85.32 h). There was considerably more variation in the MHR than is usually apparent in short-term experiments. The change in character of this trace at a calculated O2 concentration of about 2% may indicate the onset of insect paralysis. Up until near this point, the fall-off in MHR with decrease in O2 concentration was more rapid than expected from the curve in Fig. 2, which is for atmospheres of O2+N2 with minimal CO2. At this point, the difference is less marked. From this point on, the MHR decreased more quickly until it approached a value close to zero at calculated O2 concentrations of a few tenths of a percent. The MHR remained on zero, within the experimental accuracy, until the end of the run. The atmosphere in the ampoule was then equilibrated with that of the laboratory for 25 min before determining the MHR, which was 50% of the initial value at the start of the run. All six insects were alive and had lost 2.5% of their original total weight of 11.95 mg. Assuming all the products were lost during the equilibration with the laboratory atmosphere at the end of the run, the measured weight loss of the insects of 0.30 mg (precision $0.05 mg) was in accord with the calculated weight loss for the consumption of metabolic substrate of 0.27 mg based on the total measured heat of 4.482 J. The 0.15 mg of co-produced metabolic water was several times that needed to saturate the atmosphere of the closed ampoule. Arlian (1979) reported that standardised adult S. oryzae held without nutrition or water for 96 h lost 9.4% of their body weight (3.3% in the rst 72 h of fasting, and 6.1% in the last 24 h). Those insects would not have had their metabolism limited by the supply of air, unlike the present experiment. In the next section, results are given for experiments that were run for similar times in introduced anoxic CAs. 3.2.2. Sitophilus oryzae adults in anoxic atmospheres for extended times These results are summarised in Table 1, including the life status of the insects immediately after removal from the calorimeter, and after storage for 3 days in ventilated glass ampoules containing wheat grains. In all these trials the MHR during the time of exposure to the anoxic atmosphere was zero within the experimental error of about 2 mW and at the end of the experiment the MHR recovered to more than 50% of the initial

value in air. Nearly all the insects survived for at least 3 days after the anoxic treatment. The hypercarbic CA did not seem to be any more toxic than the 100% N2 CA at this temperature although the MHR recovery for the 24 h experiment was lower (61% cf. $100%). Hence, S. oryzae is tolerant of anoxic atmospheres for much longer than M. persicae (cf. Downes et al., 2003). The metabolic status under anoxic conditions was further examined using the calorimetric method to determine the ux of CO2. Seven adult S. oryzae with a fresh weight of 13.61 mg had a MHR in air of 120 mW and, after the addition of the vial of NaOH solution, the total heat rate was 153 mW. The 33 (uncertainty 724) mW was due to the ux of CO2 for metabolism in air, and the corresponding value in 100% N2, constant over the total observation period of 70 min, was 6 (uncertainty724) mW. Thus, the CO2 ux (5.5 1011 mol s1) under anoxic conditions was 18% of that in air, suggesting there was some anaerobic metabolism (see Section 4). 3.2.3. Sitophilus oryzae adults in hypoxic atmospheres As anoxic CAs were not effective in the short term (64 h) at 25 1C, hypoxic CAs supporting low rates of metabolism were investigated as being potentially more insecticidal. The MHR in air was measured before ushing the ampoule for 10 min with 1% O2+N2. The ampoule was ushed again after 3.76 h. This cycle was repeated for a total run time of about 91 h. After the rst ushing with the CA, the initial MHR was 40 mW, which corresponded to 35% of the value in air at the start of the run (cf. Fig. 2). Over the rst 3.16 h in the CA, the MHR decreased linearly to zero, which is in accord with the amount of O2 present at the start. Thus, for the last part of the rst exposure to the CA the MHR was zero in an atmosphere of zero, or near zero, O2 concentration. An interesting observation is that after thermal equilibrium was established following the second ushing with the CA, the MHR stayed at zero. Apart from thermal excursions resulting from ushing with CA, the trace indicated zero MHR until the CA was replaced with air at the end of the run, when it was 52% of the original value in air. All six insects were alive at the end of the run but within 24 h, ve were dead. In a second experiment, the same conditions as above were used except that the period between refreshing with CA was reduced to 1.04 h and the total run time was 18.7 h. At the start of

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Table 1 Recovery of metabolic heat rate in air at the end of the run, expressed as a percentage of the initial value in air at the start of the run, for adult Sitophilus oryzae after periods in anoxic atmospheres Run number Atmosphere Time in atmosphere (h) Recovery of MHR (%) Life status End of run 1 2 3 4 5 100% N2 100% N2 100% N2 60% CO2+N2 60% CO2+N2 4 20 64 24 64 $100 $100 60 61 67 8 6 6 6 6 alive alive alive alive alive After 3 days 7 6 5 6 5 alive, 1 dead alive alive, 1 dead alive alive, 1 dead

Life status was determined at the end of the run and after an additional 3 days.

Table 2 Temperatures of maximum values of metabolic heat rates, Tmax, for adult Sitophilus oryzae and Tribolium confusum, from scanning calorimetry experiments with a nominal scan rate of 1 1C min1, except for Run 2 which had a nominal scan rate of 0.5 1C min1

100 0 Heat rate (W) -100 -200 -300 -400 25 30 35 40 45 Temperature (C) 50 55 60

Run number 1 2 3 4 5 6 7

Species S. S. T. S. T. S. T. oryzae oryzae confusum oryzae confusum oryzae confusum

Atmosphere Air Air Air Air Air 5% O2+60% CO2+N2 5% O2+60% CO2+N2

Tmax (1C) 48 42 52 48 51 50 51

the rst period in the CA, the MHR was 30 mW, 40% of the original value in air. After 1 h, this had decreased linearly to about 20 mW. After refreshing with CA, nearly all this decrease was regained at the start of the second period in the CA. This sequence was repeated for the remainder of the run except that the regain after refreshing varied a little, the largest variation being after the fth ushing with CA when the initial MHR was about 40 mW. At no time during the run did the MHR for the insects in the CA fall below 20 mW (27% of the original value in air). The MHR in air at the end was 53% of the original value in air at the start of the run. All four insects were alive and vigorous at the end of the run. Unfortunately, their life status after this was not recorded.

Fig. 4. Plot of heat rates in a temperature scanning experiment, using adult Sitophilus oryzae, to determine Tmax, supposed to be the lethal temperature (see text).

3.3. Scanning experiments to determine lethal temperatures for adult Sitophilus oryzae and Tribolium confusum A representative plot (Run 4 of Table 2) of the difference between two scans as a function of temperature is given in Fig. 4. Here the intersection of the trace with the ordinate has been adjusted to correspond with the MHR at 25 1C determined isothermally before the start of the scan (Hansen and Criddle, 1990). The high temperature end of the curve was not similarly adjusted because the required isothermal measurements gave rapidly changing values under these conditions. Note that the apparent temperature coefcient for the MHR in Fig. 4 differed from that in Fig. 1a indicating that the value may depend on the temperature regime used. A more realistic comparison would have been possible if the scan had been stopped at 45 1C and an isothermal MHR determined at that temperature to better locate the high temperature end of the scan, but this would have interfered with the remainder of the scan. The natures of the two scans (not shown) used to construct the difference plotted in Fig. 4 were consistent with the insects dying during the rst scan; the second scan was featureless and akin to a scan with the ampoule empty, whereas the rst scan initially had

variations characteristic of living insects and increasingly diverged from the second scan up to about 50 1C. After this, the rst scan was smoother and the difference began to decrease until the scan traces crossed at about 54 1C. This last feature would result from the loss of water from the insects, an endothermic process likely to become more prominent, the higher the temperature. For these sealed-ampoule experiments, the amount of water evaporated would have been limited by the temperature-dependent saturated vapour pressure of water. An endothermic peak similar to that at about 44 1C shown in Fig. 4 was present in some, but not all, of the other scans. The peak can be attributed to an episodic loss of water. The area of this peak was equivalent to about 4.9 mJ and corresponded to the evaporation of about 2.0 103 mg of water, 0.03% of the fresh weight of the insects. In some experiments, the MHR was determined isothermally at 25 1C immediately after the rst scan, in the atmosphere used in that experiment, for comparison with the initial value. In all four cases checked there was some MHR, but as it was decreasing very rapidly it was not possible to quantify it. Thus, the second scan would have included some small contribution from an apparent MHR. This, and the lack of data to x the high-temperature end of the scan to isothermal results, indicated that the results in Fig. 4 would not be quantitative. However, the qualitative features of the trace may be useful to determine the temperature at which the insects died. The obvious candidate is the temperature of maximum MHR, dened by the intersection of extensions of the two arms of the curve, denoted as Tmax and listed in Table 2. The slower scan used in Run 2 than that in Run 1 resulted in a signicantly lower Tmax. This is in accord with the data listed by

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Fields (1992), in which longer exposure times resulted in mortality at lower temperatures. In our short-term experiments, the 5% O2+60% CO2+N2 CA did not have a marked effect on Tmax relative to the values in air. The results in Fig. 1a show that this CA did have an effect at longer exposure times.

7 6 Heat flow (mW) 5 4 3 2 1 40

Experimental Fitted Calculated

3.4. Combined thermo-gravimetric and differential thermal-analysis experiment on adult Sitophilus oryzae To test whether the upward trend of the plot in Fig. 4 does have a large component due to the endothermic loss of water, thermogravimetric/differential thermal-analysis experiments were done using adult S. oryzae at a nominal scan rate of 1 1C min1. These experiments were undertaken with owing air in the instrument (cf. the sealed ampoules for the experiments in the last section). Fig. 5 shows that the signicant loss in weight of the four adult S. oryzae (initial weight 7.68 mg) did not start until about 55 1C, so it is not clear that loss of water was the cause of the upward trend in Fig. 4. (In this context, note the difference in scale of the ordinates in Figs. 4 and 5.) However, it is possible that thermal lag had not been fully accounted for in the instrument. To check this, a run was done using high purity tetracosane, C24H50 (Koch-Light Laboratories), which has a melting point of 51.1 1C. The start of melting was close to the stated melting point, so thermal lag was not signicant. The loss in weight during the insect experiment was 49.9%, in good agreement with Arlians (1979) determination of the water content of adult S. oryzae of 50.574.1% (n 17). It was expected that much of the heat ow recorded for the insects in the DTA experiment would be associated with the heat of vapourisation of water or other volatiles. The asymmetric DTA peak was deconvoluted to produce the three overlapping peaks, shown in Fig. 6, which have areas in an approximate ratio of 10:30:60. The TG plot was then differentiated with respect to time (note, not temperature) and the rate of weight loss was converted to a heat rate using the temperature-dependent heat of vapourisation of water (Hodgman, 1960). The resultant curve, scaled by the factor 0.77 and the ordinate adjusted by 1.3, is shown overlaid on the DTA plot in Fig. 6.

50

60

70 80 Temperature (C)

90

100

Fig. 6. Deconvolution of the differential thermal-analysis (DTA) results for adult Sitophilus oryzae given in Fig. 5 overlaid with a scaled plot (see text) of the weight (thermo-gravimetric results) differentiated with respect to time and converted to heat ow (labelled calculated). The triangles are interpolated points from the experimental DTA results in Fig. 5. The tted solid line is the sum of the three smaller peaks.

4. Discussion 4.1. Effect of controlled atmospheres and temperature The temperature dependence of the MHR in air was quite different for adult and larval S. oryzae (Fig. 1a and b). In these
8.0 7 6 Heat flow (mW) 5 4 5.5 3 2 1 40 50 60 70 Temperature (C)
Fig. 5. Combined thermo-gravimetric (TG) and differential thermal-analysis (DTA) results in owing air for four adult Sitophilus oryzae, with total weight at start of 7.68 mg and nominal scan rate of 1 1C min1.

Insect heat flow

7.5 7.0 Weight (mg) 6.5 6.0

5.0 Insect weight 4.5 4.0 80 90 100

short-term experiments, the adults survived the nal step of 30 min at 45 1C, but the later scanning experiments, in which the lethal temperature appeared to be 48 1C, combined with data in the literature, suggest longer times at this temperature would be lethal. The temperature dependence of the MHR (Fig. 1a) for the adult S. oryzae is almost certainly more representative of the longer-term behaviour of the insects at a given temperature than that from the scanning experiment because of the different times it took to increase the temperature to 45 1C. The pauses in the stepped-isothermal experiment, although relatively short, would permit some acclimatisation. The temperature dependence for the MHR of the larvae in the wheat grains strongly suggests they did not survive much above 30 1C and that they are more susceptible than the adults to moderate increases in temperature. Acclimatisation may be important in natural habitats. For both the adult and larval stages of S. oryzae, the presence of 60% CO2 enhanced the effect on the MHR of the increase in temperature to 45 1C for the CA with 5% O2+60% CO2+N2, relative to the CA with 5% O2+N2. At least in the case of the adults, and almost certainly also for the larvae, death resulted from this short exposure at 45 1C. There was sufcient O2 to maintain a high level of metabolism, but the high CO2 concentration nevertheless resulted in the MHR declining rapidly from the value plotted in Fig. 1a, and death of the insects. The effect on the MHR at 45 1C of the presence of 60% CO2 in the 5% O2+60% CO2+N2 CA was less marked for T. confusum, with only the adults dying. In the absence of any signicant MHR in anoxic atmospheres, loss of water, an endothermic process (giving positive observed heat rates), is the probable reason for the small negative values of the apparent MHR (expressed as a percentage of the exothermic 25 1C value in air), particularly at 45 1C. This process may also have occurred for insects that survived at 45 1C, though to a much smaller extent, but it was masked by a signicant MHR. The longterm experiments at 25 1C using S. oryzae adults in anoxic atmospheres showed that these CAs per se are not insecticidal for time periods equivalent to those required for the experiments of Fig. 1ad. However, when their metabolism was largely, or completely, switched off under anoxic conditions, neither species survived a temperature of 45 1C. As experiments were not done at 45 1C using a 100% N2 atmosphere, it is not possible to assess the

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importance of the presence of the CO2 in the 60% CO2+N2 atmosphere. Hoback and Stanley (2001) discussed the evidence for anaerobic pathways in terrestrial insects. For all the species we have examined, the MHR under anoxic conditions was less than about 1% of the aerobic value. This indicates that the anaerobic metabolic rate was low, or that the reactions of the metabolic pathway had small associated heat effects. From a consideration of literature results, Hoback and Stanley concluded that anoxia can inuence a broad spectrum of metabolic and developmental events by inhibiting key aerobic pathways. They cited results for adults of the esh y, Sarcophaga crassipalpis Macquart, which under normal circumstances can be induced to tolerate a high temperature of 45 1C by brief exposure to elevated, but sublethal, temperatures. However, brief exposure of 30 min to anoxia inhibited the development of this thermal tolerance. To summarise, in these short-term trials the presence of 60% CO2 in a CA containing 5% O2, or an anoxic CA, greatly improved the effectiveness of the 45 1C treatment for killing adult S. oryzae, while larvae probably did not survive in any atmosphere much above 30 1C. Adult T. confusum followed a similar pattern to adult S. oryzae, but were somewhat more susceptible. Tribolium confusum larvae were more resistant with only the anoxic CA containing 60% CO2 being effective.

that there was signicant anaerobic metabolism needs to be treated with caution. There could be sources of the CO2 other than directly from metabolism. For instance, Wigglesworth (1972) notes that, at least for some species, under anoxic conditions the haemolymph can increase in acidity resulting in the liberation of CO2 from bicarbonate in the haemolymph and tissues. If there was metabolism under anoxic conditions then it was associated with a very small MHR (magnitude o2 mW). The results in Fig. 2 show that the MHR of adult S. oryzae in 1% O2+N2 at 25 1C was about 35% of the value in air. The long-term experiment showed that the metabolism could be continued at about that rate for at least 18 h. However, should the MHR fall to around zero, as a result of low oxygen concentrations, then an atmosphere of 1% O2+N2 was insufcient to restart the metabolism, or at least any metabolism that produced heat at a rate of more than about 1% of the normal value in air. Additional experiments are required to determine whether O2 concentrations of less than that of air are sufcient for the MHR to resume and whether other factors are important, such as the length of time over which the MHR has been close to zero. A more detailed study of the effects of hypoxia by microcalorimetry would provide a useful background for biochemical investigations of the basic processes involved in the switching on again of the metabolism. 4.3. Scanning experiments to determine lethal temperatures for adult Sitophilus oryzae and Tribolium confusum This appears to be a simple technique but a more extensive set of experiments would be useful to conrm the initial promising results. This might include experiments that are terminated at temperatures on either side of the indicated Tmax to conrm the insects life status. Oxygen depletion of the atmosphere in the ampoule needs to be considered. For example, in Run 6 of Table 2 it can be calculated that about 40% of the oxygen in the ampoule was consumed by the time the temperature had reached 50 1C, whereas only about 8% had been consumed by 50 1C for an experiment run in air (Run 4 of Table 2, and Fig. 4). As the change in slope at Tmax is quite marked, fewer insects in the sample would sufce and O2 depletion effects would be reduced. If slow heating rates are of interest then the atmosphere can be replenished automatically on any desired schedule. The mortality studies at elevated temperature for adult S. oryzae listed by Fields (1992) cover a variety of experimental conditions, making comparison with the Tmax values difcult. Overall, they perhaps indicate mortality temperatures a few degrees higher than the Tmax values. Of the mortality studies listed by Fields for adult T. confusum, that by Oosthuizen (1935) contains data most suitable for comparison with our Tmax values. The times for 50% mortality at temperatures of 44, 46, 48 and 50 1C are given as 7 h, 1.2 h, 26 min and 4.9 min, respectively. Oosthuizen also noted a wide range of variation in resistance to high temperature within a given population of T. confusum. Given these uncertainties, it seems likely that the Tmax temperatures do indeed represent the lethal temperatures. 4.4. Combined thermo-gravimetric and differential thermal-analysis experiment on adult Sitophilus oryzae A tentative classication can be made of the three peaks arising from the deconvolution of the DTA data. The smallest peak starts at a temperature near that at which death of the insects occurs. In a preliminary examination of the data, there were indications that the thermal effect arising from the weight loss, presumably due to loss of water from the gut, was less than sufcient to account for the size of this peak. It was envisaged that

4.2. Effect of hypoxic and anoxic atmospheres The MHR for adult S. oryzae at 25 1C (Fig. 2) declined less rapidly with decrease in O2 concentration than that for M. persicae at 40 1C (Downes et al., 2003). The MHR was reduced to 50% of the value in air at O2 concentrations of 1.8% and 6.3% for S. oryzae and M. persicae, respectively. Emekci et al. (2002) found a similar tolerance of low O2 levels by T. castaneum at 30 1C. They found that the respiration rate, as measured by the production of CO2, was 34% greater in 5% O2+N2 than in 21% O2+N2 (see below for further comment), but that it was much smaller at O2 concentrations of 1% and 2% O2+N2. This higher tolerance by granary insects of low O2 levels may have developed since grains were rst stored, or else is a reection of their former habitat. Hoback and Stanley (2001) consider that insects that feed on stored grain encounter hypoxia because O2 is depleted to less than 2%, which they believe is due to the respiration of the grain, when storage is airtight. The calculated values for O2 and CO2 concentrations in Fig. 3 for the hermetic storage simulation require some comment. Respiration is usually measured as the rate of O2 consumption, RO2, or the rate of CO2 production, RCO2, both in mol s1, and less often as the MHR, F, in mW. For fully aerobic respiration with a carbohydrate type of substrate such as glycogen, RCO2/RO2 1, and F/RO2 455715 kJ mol1 of O2, as noted above. Downes et al. (2003) found with M. persicae, at 40 1C in O2 concentrations near that of air, F/RO2 429754 kJ mol1 and RCO2/RO2 0.9870.15, which are in good agreement with expected values. For O2 concentrations near 4%, F/RO2 465737 kJ mol1, again in good agreement, but RCO2/RO2 1.3270.15. If adult S. oryzae follow this same trend then the calculated values of the O2 concentrations in Fig. 3 should be reasonably accurate, but the CO2 concentrations will be too low in atmospheres with low O2 concentrations. Further support for this conclusion comes from Emekci et al. (2002) who have discussed their ndings, and those of others, of RCO2/RO241 at low oxygen concentrations. Another factor that would introduce error in the calculated O2 and CO2 values would be a change in nutrition type from carbohydrate to protein or lipid. In view of the large uncertainties associated with the relatively small heat rate from which the CO2 ux was derived, the nding

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contributions to this peak could arise from endothermic reactions that do not involve signicant loss of weight, including processes such as melting of waxes and storage lipids, melting of cell membranes, and thermal denaturation of proteins. The middle peak is, presumably, mainly due to loss of water from the haemolymph, so using the value above for the total water content it can be calculated the haemolymph amounts to 15% of the fresh weight of the insects. This value is similar to those listed by Hadley (1994) for a range of insects. He also discusses the transport of water between the haemolymph and tissues in response to dehydration, but these considerations are unlikely to be important given the time-scale of the present TG/DTA experiment. The largest peak is presumably associated with loss of intracellular water. Ever-increasing concentrations of simple solutes during dehydration probably have a minor effect on the specic heat of vapourisation, as shown by the data for salts (Horvath, 1985), but there is a considerable literature (see, for example, Drost-Hansen and Clegg, 1979) postulating the existence of bound or vicinal water, which is assumed to have a higher heat of vapourisation than normal water. In summary, although all the above thermal effects may be operative, given the adjustments made to the calculated curve, it is believed that they cannot be identied from the present data. Nonetheless, the three peaks in the data can be explained largely as the progressive loss of water from the gut, haemolymph and intracellular regions as temperature was increased.

for adult S. oryzae, such as lipid melting and protein denaturation, was sought using a combined TG-DTA technique, but could not be identied with the available precision. However, it was observed that on increasing the temperature three types of water were evolved progressively near and above the lethal temperature which were assigned as being from the gut, haemolymph and intracellular regions, respectively. The results for each of the above methods are not exhaustive, and they may be species-specic. However, the purpose of this paper will have been achieved if a wider audience appreciates the potential of calorimetry to contribute, alongside other methods, to devise treatments for the control of stored-product insects.

Acknowledgements This research was funded by the Foundation for Research, Science and Technology. Thanks are due to Dr. P.G. Fields for helpful correspondence, Dr. B. Howlett for reviewing the manuscript, D. Park for editorial assistance, and Dr. B. Champ and an anonymous reviewer for suggesting improvements to an earlier draft. References
Arlian, L.G., 1979. Signicance of passive sorption of atmospheric water vapor and feeding in water balance of the rice weevil, Sitophilus oryzae. Comparative Biochemistry and Physiology 62A, 725733. Criddle, R.S., Hansen, L.D., 1999. Calorimetric methods for analysis of plant metabolism. In: Kemp, R.B. (Ed.), Handbook of Thermal Analysis and Calorimetry, vol. 4. Elsevier, Amsterdam, pp. 711763. Dean, G.A., 1911. Heat as a means of controlling mill insects. Journal of Economic Entomology 4, 142158. Donahaye, E.J., Navarro, S., Rinder, M., Azrieli, A., 1996. The combined inuence of temperature and modied atmospheres on Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Journal of Stored Products Research 32, 225232. Downes, C.J., Carpenter, A., Hansen, L.D., Lill, R.E., 2003. Microcalorimetric and mass spectrometric methods for determining the effects of controlled atmospheres on insect metabolism. Thermochemica Acta 397, 1929. Drost-Hansen, W., Clegg, J.S. (Eds.), 1979. Cell-associated water. In: Proceedings of a Workshop on Cell-Associated Water, Boston, MA, September 1976. Academic Press, New York. Emekci, M., Navarro, S., Donahaye, E., Rinder, M., Azrieli, A., 2002. Respiration of Tribolium castaneum (Herbst) at reduced oxygen concentrations. Journal of Stored Products Research 38, 413423. Fields, P.G., 1992. The control of stored-product insects and mites with extreme temperatures. Journal of Stored Products Research 28, 89118. Glasstone, S., 1946. Textbook of Physical Chemistry, second ed. D. van Nostrand Company, New York. Hadley, N.F., 1994. Water Relations of Terrestrial Arthropods. Academic Press, San Diego. Hansen, L.D., Criddle, R.S., 1990. Determination of phase changes and metabolic rates in plant tissues as a function of temperature by heat conduction DSC. Thermochimica Acta 160, 173192. Hoback, W.W., Stanley, D.W., 2001. Insects in hypoxia. Journal of Insect Physiology 47, 533542. Hodgman, C.D. (Ed.), 1960. Handbook of Chemistry and Physics, 42nd ed. The Chemical Rubber Publishing Co., Cleveland. Horvath, A.L., 1985. Aqueous Electrolyte Solutions. Ellis Horward Limited, Chichester. Mahroof, R., Subramanyam, B., Eustace, D., 2003. Temperature and relative humidity proles during treatment of mills and its efcacy against Tribolium castaneum (Herbst) life stages. Journal of Stored Products Research 39, 555569. Oosthuizen, M.J., 1935. The effect of high temperature on the confused our beetle. Minnesota Technical Bulletin 107, 145. Roesli, R., Subramanyam, B., Fairchild, F.J., Behnke, K.C., 2003. Trap catches of stored-product insects before and after heat treatment in a pilot feed mill. Journal of Stored Products Research 39, 521540. Soderstrom, E.L., Brandl, D.G., Mackey, B., 1992. High temperature combined with carbon dioxide enriched or reduced oxygen atmospheres for control of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Journal of Stored Products Research 28, 235238. Wigglesworth, V.B., 1972. The Principles of Insect Physiology, seventh ed. Chapman and Hall, London.

5. Conclusions The proposal in the introduction that heat treatments for disinfestation performed in a hypoxic or hypercarbic CA may be effective at lower temperatures than required in air was supported by the results. Thus, heat treatment at 45 1C was more effective when the CA was 5% O2+60% CO2+N2, or 60% CO2+N2, than in air, but larval T. confusum still survived in the former CA, indicating there were differences in the response between species and/or stage. The results of these short-term experiments provide a basis for pilot-scale trials. In more detailed investigations, the results of which are summarised below, calorimetric methods were developed to delineate the response of insects to changes in conditions of life-threatening proportions. The MHR of adult S. oryzae was changed little over a wide range of oxygen concentrations, the value in air was close to that in pure oxygen and was only reduced by about 20% and 50% at oxygen concentrations of 5% and 2%, respectively. The response arising from hermetic storage was readily identied by measuring the MHR as a function of time. Judging by the change in character of the trace, paralysis occurred at about 50% of the initial MHR value in air, at a calculated oxygen concentration of about 2%. A simple chemical model of the metabolism gave a time for oxygen depletion which corresponded to that observed for the cessation of metabolic heat production, and a weight loss for the insects, calculated using the total metabolic heat, in accord with the observed value. In other experiments, production of metabolic heat ceased in introduced anoxic atmospheres but could be restarted on returning to air, even after several days of anoxia. Metabolism continued at a decreased rate on going from air to low oxygen levels ($1%), which were below that required to restart metabolism after anoxia. Lethal temperatures for adult S. oryzae and T. confusum were readily determined using the calorimeter in scanning mode. Evidence of thermochemical events near the lethal temperature