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Respiration of Tribolium castaneum (Herbst) at reduced oxygen concentrations
Journal of Stored Products Research 38 (2002) 413–425
Respiration of Tribolium castaneum (Herbst) at reduced oxygen concentrations M. Emekcia, S. Navarrob,*, E. Donahayeb, M. Rindnerb, A. Azrielia b
a Plant Protection Department, Faculty of Agriculture, University of Ankara, 06110 Ankara, Turkey Department of Stored Products, The Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel
Accepted 17 August 2001
Abstract Adults, eggs, young and old larvae and pupae of Tribolium castaneum (Herbst) were exposed to atmospheres containing 1%, 2%, 3%, 5%, 10%, and 15% oxygen in nitrogen at 301C and 70% r.h. Respiration rates were determined with a gas chromatograph. The oxygen intake and carbon dioxide output by insects were expressed in ml/insect/h or ml/mg/h. Adults exposed to 21% oxygen required an initial acclimatization period of at least 5 h, after which the respiration rate remained stable. Based on this finding, all the respiration measurements were carried out after an initial adaptation of insects to the respirometer conditions for 24 h. Respiration of eggs, young and old larvae, pupae, and adults at 301C in normal atmospheric air was at rates of 0.0121, 9.25, 8.45, 1.45, and 4.67 ml CO2/insect/h, respectively. Respiration rates of the same stages in terms of insect weight were 0.32, 29.08, 3.33, 0.59 and 2.37 ml CO2/mg insect/h, respectively. At reduced oxygen levels respiration rates of eggs, larvae and pupae were proportional to the oxygen levels. Adult respiration was higher for 3% and 5% oxygen than for normal atmospheric air with rates of 4.77 and 4.98 ml CO2/insect/h, respectively. In adults, RQ values for the same oxygen levels were also higher than for normal atmospheric oxygen and were 1.07 and 1.18, respectively. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Tribolium castaneum; Development stages; Respiration; Reduced oxygen
1. Introduction Modified atmosphere (MA) technology has been widely recognized as a means of preserving stored grain (Bailey and Banks, 1975; Banks et al., 1980; Navarro et al., 1979; Navarro and *Corresponding author. Fax: +972-3-9604428. E-mail address: [email protected] (S. Navarro). 0022-474X/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 4 7 4 X ( 0 1 ) 0 0 0 4 5 - 5
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Donahaye, 1990). Among the MA application methods, the bio-generation of the atmosphere through the respiration of insect pests and microorganisms has been shown to be effective in depleting the oxygen (O2) and increasing the carbon dioxide (CO2) concentration of the intergranular space. Using this process in hermetic storage, it is possible to arrest insect pest development and to minimize storage damage (Navarro et al., 1993). Improvements in gas generation technology can also reduce the cost of MA generation. Such improvements together with the laboratory experiments to demonstrate the effectiveness of low O2 or high CO2 concentrations could lead to a more widespread use of MAs in the future (Annis and Dowsett, 1993). Most of the research on MAs has naturally been concentrated on the effectiveness of different atmospheric gas combinations as a means of insect control. However, the effects of sublethal concentrations of MAs on insects, such as delaying development, impaired metamorphosis and altered fecundity, have not been well documented (Bailey and Banks, 1980). Among the effects of MAs, their influence on insect respiration has also not been sufficiently studied. Knowledge of the respiration rate of insects in storage systems is important, since this information can be utilized in a number of situations such as in calculating its effect on heating of bulk-stored grain. Respiration is also a good index of the physiological responses of the insects to the environment in which they are exposed. Several works have been carried out on the respiratory mechanisms of stored product insect pests, mostly at normal atmospheric air. Park (1936) investigated O2 consumption of Tribolium spp. in relation to body weight, sex, and food medium. Birch (1947) studied the O2 consumption of various developmental stages of S. oryzae (L.), and Rhyzopertha dominica (F.) over a range of temperatures and at different relative humidities (r.h.). They showed that O2 consumption increased as the development proceeded up to the prepupal stage. Calderwood (1961) conducted studies on the O2 consumption of Tribolium confusum du Val. and Tribolium castaneum in relation to the temperatures between 151C and 371C, and found a positive correlation between metabolic rate and temperature. Chaudry and Kapoor (1967) investigated the respiratory metabolism of T. castaneum in relation to temperature. Carlson (1966, 1968a, b) measured the respiration of T. confusum under different CO2:O2 ratios and reported that sublethal exposure to CO2 or N2 reduced the O2 consumption in adults. Dumas et al. (1969) investigated the larval respiration of Tenebroides mauritanicus (L.). They reported that both O2 consumption and CO2 production were progressively reduced as the pressure was lowered from 200 to 35 mmHg. Aliniazee (1971) showed that high CO2 levels (8–16%) severely suppressed respiration in T. confusum. Navarro and Calderon (1979) investigated the effects of low atmospheric pressures and low O2 concentrations on pupal respiration, mortality, and weight loss in Ephestia cautella (Walker), and stated that respiration was suppressed in both types of atmospheres at an O2 concentration equivalent to 3%. Campbell and Sinha (1978), studying the bio-energetics of Cryptolestes ferrugineus (Stephens), stated that prepupae, pupae, and adults had a lower respiration rate than did the developing larvae. White and Sinha (1981), and Campbell and Sinha (1990) reached the same conclusion in Oryzaephilus surinamensis (L.) and C. ferrugineus, respectively. Donahaye (1992) investigated the metabolic rate in terms of O2 uptake, CO2 production, and losses in wet and dry weights in adults of T. castaneum which were exposed to two kinds of MAs. He found that respiration was higher in the unselected strain as compared with both the high carbon dioxide concentration (HCC), and low oxygen concentration (LOC) selected strains at 261C and 95% r.h. Many laboratory studies
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have concentrated on the effectiveness of atmospheres containing 0–1% O2 particularly on the control of the adult stage of insects, whereas, atmospheres of 1–5% O2 concentrations are easier to obtain and maintain economically in sealed storages for the application of MAs (Annis and Dowsett, 1993). There is a lack of information on the effect of low O2 levels, particularly those higher than 1%, on the different developmental stages of stored grain insects in terms of both lethal and sublethal aspects. Such information can be particularly useful when considering simulation models. The objective of this work was to study the respiration rates of the development stages of T. castaneum at low levels of O2 which prevail when atmospheres under gas tight conditions are modified by the metabolic activity of insects.
2. Materials and methods 2.1. Insect culture techniques T. castaneum eggs were obtained from 1000 to 2000 young adults placed in 1-l jars that contained approximately 250 g of flour and 5% yeast (by weight). Every three days the jars were sieved for egg removal. These adults were used for oviposition for up to one month. Approximately 2000–3000 three day old eggs were placed in jars, containing B500 g finely ground wheat and 5% yeast. The medium was later sieved to obtain young larvae, mature larvae, pupae and adults (Donahaye, 1990). 2.2. Modified atmospheres The respiration rate of T. castaneum was measured in atmospheres containing 1%, 2%, 3%, 5%, 10% or 15% O2 in N2 at 301C. Normal air served as a control. Gas mixtures were prepared as described by Donahaye (1990), and maintained at a flow rate of 15 ml/min at 70% r.h. (Navarro and Donahaye, 1972). Table 1 shows the different development stages, the number of individuals and age groups of T. castaneum used in studying respiration rates at low oxygen concentrations. Table 1 Numbers and the ages of Tribolium castaneum at the stages used for testing their respiration rates Development stage
Number of insects per treatment
Age of insectsa (days)
Eggs Young larvae Old larvae Pupae Adults
300 100 100 100 100
0–1 7–10 18–22 0–1 10–14
a Age for larval stages from egg stage; pupae from pupation; and adults from emergence.
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2.3. Exposure flasks The flasks consisted of 50–60 ml bottles with 2 cm wide necks. Their volumes were measured carefully before the experiments. The bottles were equipped with silicon septa and aluminum lids. A crimper was used to seal the bottles tightly. Two N.14 syringe needles of 0.65 32 mm2 were inserted through the septa. One served as an inlet for the gas mixture while the other served as an outlet.
2.4. Exposure techniques Known numbers of insects (Table 1) were weighed and transferred to the B60 ml experimental bottles containing 200 mg flour for all development stages, except eggs and pupae, which did not need the food. After closing, the bottles were connected to the exposure apparatus. A soap-bubble flow meter was attached to the outlet needle of each bottle to verify the flow rate through the bottle and to ensure that there were no blockages in the system. The bottles were held at 301C in a thermostatically controlled room. Following 24 h of exposure (for all stages except eggs), the air composition within the bottle was measured by sampling with a 1 ml ‘‘pressure-lock’’ syringe through the silicon septum. The inlet and outlet ports of the bottles were then blocked tightly for approximately 1 h (24 h for eggs) by means of metal clamps. At the end of this period flasks were sampled as described previously. Gas concentrations were determined using a Tracer 565 gas chromatograph equipped with twin thermal conductivity cells, dual columns (each 40 1/1600 i d), and a sample loop thermostatically regulated at 501C. Column 1 was packed with ‘‘Porapak Q’’, while column 2 was packed with ‘‘molecular sieve 5a’’. Percentage concentration of the gas components was computed by a Spectra Physics SP 4100 integrator. Preliminary experiments at a normal atmospheric air pressure showed that adults had to be exposed for acclimatization for not o5 h prior to experimentation (Fig. 1). All stages except eggs were, therefore, exposed for a 24 h acclimatization period before gas sampling.
Carbon dioxide production
6.0
5.0
µl CO 2 /insect/h
4.0
3.0 µl CO 2 /mg/h 2.0
1.0 0
8
16
24
32
40
48
Time (h)
Fig. 1. Change in respiration rate of Tribolium castaneum adults during acclimatization in normal atmospheric air at 301C and 70% r.h.
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The possibility of leakage due to successive sampling from the small volume of experimental flasks (50–60 ml) with a relatively large sample sizes (1 ml) was investigated. Although we did not find any statistical difference between the first two successive samples taken from the same flasks which were in the gas tight position, the method was not suitable for the detection of the tiny O2 consumption of eggs exposed to low oxygen concentrations. It was suspected that slight leakage occurred. In order to avoid the effects of possible leakages, results for eggs were expressed on the basis of CO2 production instead of O2 uptake. Since one of the aims of this study was to provide more information on respiration we preferred to give the CO2 production per mg body weight per h together with the CO2 production per insect per h in each figure for the different developmental stages. Such information is generally lacking in the literature.
3. Results Results showed that the respiration rate of immature stages increased with the increase of O2 levels. The most pronounced suppressant effect of low O2 concentrations on respiration was observed with eggs (Fig. 2) and young larvae (Fig. 3). For these developmental stages, CO2 production was lower at concentrations of up to 3% O2 as compared to the controls. At concentrations of 1–15% O2, respiration of old larvae appeared higher than that of young larvae (Fig. 4). However, examination of the respiration rate by body weight reveals that young larvae respired much more rapidly than old larvae (Figs. 3 and 4). A further increase of O2 level to 21% leads to even higher respiration in young larvae. As development proceeded, respiration was markedly suppressed when O2 was lowered to 1% in old larvae and to some extent in pupae (Figs. 4 and 5). RQ values in normal atmospheric air were 1.02 and 0.72 for old larvae and pupae, respectively (Table 2.). Unlike prepupal stages, in adults high CO2 production was obtained at 3–10% O2 (Fig. 6); but adult respiration was less affected by the experimental range of O2 concentrations than that of younger larvae or eggs. The RQ value in normal air for adults was 0.89 (Table 2).
Carbon dioxide production
0.35 0.30 0.25
µl CO 2 /mg/h
0.20
µl CO 2 /10 eggs/h
0.15 0.10 0.05 0.00 0
5
10 15 Oxygen concentration (%)
20
Fig. 2. Respiration rates of Tribolium castaneum eggs in low oxygen concentrations in nitrogen at 301C and 70% r.h.
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Carbon dioxide production
28 24 µl CO 2 /mg/h
20 16
µl CO2 /insect/h
12 8 4
0
5
10 15 Oxygen concentration (%)
20
Fig. 3. Respiration rates of Tribolium castaneum young larvae in low oxygen concentrations in nitrogen at 301C and 70% r.h. 9 Carbon dioxide production
8 7
µl CO2 /insect/h
6 5
µl CO2 /mg/h
4 3 2 1 0 0
5
10 15 Oxygen concentration (%)
20
Fig. 4. Respiration rates of Tribolium castaneum old larvae in low oxygen concentrations in nitrogen at 301C and 70% r.h.
With some exceptions RQ values generally tended to increase at all life stages as the O2 concentrations decreased (Table 2).
4. Discussion 4.1. Adult respiration in air Respiration of Tribolium spp. has mostly been studied with the adult stage and at normal atmospheric air pressure. A comparison of our results for oxygen intake of adults with data from the literature is shown in Table 3 although differences in temperature, r.h. and particularly
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Carbon dioxide production
1.50
1.25 µl CO 2 /insect/h 1.00 µl CO 2 /mg/h
0.75 0.50 0.25 0
5
10 15 Oxygen concentration (%)
20
Fig. 5. Respiration rates of Tribolium castaneum pupae in low oxygen concentrations in nitrogen at 301C and 70% r.h.
Table 2 Respiratory quotients of life stages of Tribolium castaneum at 301C and 70% r.h. at various concentrations of oxygen in nitrogen O2 concentration (%)
1 2 3 5 10 15 21
Life stages Eggs
Young larvae
Old larvae
Pupae
Adults
F F F 1.85 0.96 0.92 0.83
2.40 1.05 0.95 1.04 1.00 1.00 1.01
0.93 1.12 1.06 1.03 1.00 1.06 1.02
1.07 1.16 0.98 1.18 0.78 0.64 0.72
1.22 0.92 1.07 1.18 1.03 0.96 0.88
6.0
Carbon dioxide production
5.5
µl CO 2 /insect/h
5.0 4.5 4.0 3.5 µl CO 2 /mg/h
3.0 2.5 2.0 1.5 1.0 0.5 0
5
10 15 Oxygen concentration (%)
20
Fig. 6. Respiration rates of Tribolium castaneum adults in low oxygen concentrations in nitrogen at 301C and 70% r.h.
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Table 3 Comparative data on respiration rates of Tribolium spp. adults in air from the literature Species
Temperature (1C)
Respiration ratesa ml O2/insect/h
T. confusum ‘‘ ‘‘ ‘‘ ‘‘ ‘‘ ‘‘
29 25 37 30 30
T. castaneum ‘‘ ‘‘ ‘‘ ‘‘ ‘‘ ‘‘ ‘‘
30 35 26.6 26 20 25 30 35
a
ml O2/mg/h
Source ml CO2/mg/h
1.79–2.31 1.6 1.6
2.64 2.69
Park (1936) Edwards (1935) Kennington (1957) Calderwood (1961) Calderwood (1961) Carlson (1966) Carlson (1968a)
4.26 2.15 1.19 1.68 2.37 3.31
Chaudry and Kapoor (1967) Chaudry and Kapoor (1967) Aliniazee (1971) Donahaye (1992) Present work Present work Present work Present work
3.14 5.83
1.09 2.39
2.49 3.71 5.73 7.15
1.36 1.83 2.97 3.55
Converted to ml wherever necessary.
acclimatization periods need to be taken into consideration. Park (1936) reported the O2 consumption of T. confusum in the range of 1.79–2.31 ml O2/mg/h at 281C. His studies were detailed and also included sexual differences and conditioning of food. According to Edwards (1958) T. confusum respired 1.6 ml O2/mg/h (converted from mm3/mg/h) at 261C. Similar results were obtained by Kennington (1957) at 291C. Chaudry and Kapoor (1967) obtained a lower O2 uptake by T. castaneum of 1.09 ml O2/mg/h at 301C, increasing to 2.39 ml O2/mg/h at 351C. Our results showing a respiration rate of 1.83 ml O2/mg/h at 251C and 2.97 ml O2/mg/h at 301C are in agreement with the data in the literature. Calderwood (1961) reported the O2 uptake by T. confusum adults at 251C and 371C as 3.14 ml O2/insect/h (converted from mmol/h) and 5.83 ml O2//insect/h (converted from mmol/h), respectively. In our experiment, O2 uptake was found to be 5.73 ml O2/insect/h at 301C. Aliniazee (1971) reported that CO2 production in T. castaneum at 26.61C was 4.26 ml CO2/mg/h (converted from mg/g/h). Donahaye (1992) stated that in T. castaneum, CO2 expiration was 2.15 ml CO2/mg/h (converted from mmol/mg/min) at 261C and 95% r.h. In two separate works, Carlson (1966, 1968a) reported that CO2 production in T. confusum at 301C was 2.64 ml CO2/mg/h (converted from mg/mg/min) and 2.69 ml CO2/mg/h (converted from mmol/mg/min), respectively. In these studies, the acclimatization period was reported as a maximum of 1 h (Carlson, 1966, 1968a). In the present work, we found that before acclimatization respiration rate was 2.9 ml CO2/ mg/h and after 5 h of acclimatizing insects to the experimental conditions the respiration rate was 2.0 ml CO2/mg/h (Fig. 1). The difference between the results of these studies and ours can partly be attributed to the differences in acclimatization of insects to the test conditions.
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4.2. Respiration of different developmental stages of other species at normal atmospheric pressure Birch (1947), who studied the respiration of S. oryzae at 301C and 70% r.h. at normal atmospheric air concluded that the rate of oxygen consumption per insect increased during the development from the egg to prepupal stage, then fell during pupation. In the same work, O2 consumption in S. oryzae populations composed of young larvae, old larvae, mainly pupae (84%) and 1-week-old adults was given as 5.30, 26, 5.77, and 5.40 ml/insect/h (converted from mm3/insect/h), respectively. In our experiment, we observed a similar variation in respiration rates according to life stages, but respiration was higher in young larvae than in older ones in normal air (Figs. 3 and 4). In this respect, our results were closer to those of Campbell and Sinha (1978). They stated a reduction in O2 uptake of C. ferrugineus on the basis of dry body weight from 149 ml O2/mg/h in first instar larvae to 27.5 ml O2/mg/h in the adult stage in normal atmospheric air at 301C and 70% r.h. White and Sinha (1981) reported the same respiration pattern in O. surinamensis. They found that the hourly O2 consumption per insect at 301C and 80% r.h. increased to a maximum of 108 ml, when the majority of insects were entering the second instar, decreasing to 0.4 ml at pupation and rising to 4.4 ml during the first few days of the adult stage. 4.3. Respiration at reduced O2 levels We had difficulties in comparing our findings to those in the literature due to lack of information on the respiration of T. castaneum at reduced O2 concentrations, particularly on the variations in response to different development stages. Our results showed that respiration tended to increase as the O2 concentration increased from 1% to 21% for all development stages except the adult. During the course of this work, the respiration rate was suppressed at O2 levels lower than 5% in eggs and young larvae (Figs. 2 and 3). Low O2 levels were more effective in reducing the respiration rate of young larvae than old larvae. On the other hand the CO2 production expressed per insect was higher in young larvae than in old larvae in the control (21% O2) (Figs. 3 and 4). Pupal respiration decreased as O2 concentrations dropped (Fig. 5). Similar results on respiration of E. cautella pupae were reported at reduced O2 atmospheres at 261C and 93–99% r.h. (Navarro and Calderon, 1979). Accordingly CO2 production by E. cautella was approximately 0.1, 0.2 and 0.35 ml CO2/100 mg/h at O2 concentrations of 1%, 3% and 21%, respectively. These figures were in agreement with our findings which showed relatively less disruption of the pupal stage at low O2 concentrations. One of the most interesting results in this study was the increased respiration rate of adults at 3% and 5% O2 (Fig. 6) which can be considered as a compensation response to the O2 deficiency. Donahaye (1992) provided data on the physiological differences between unselected and selected strains of adult T. castaneum for hypoxia and hypercarbia at 261C and 95% r.h. We calculated that CO2 production was reduced from 1.88 to 1.34 ml CO2/mg/h (converted from mmol/mg/min) for the unselected strain when respiration was measured initially and after exposure for 24 h to 0.5% O2 in N2. His results, though recorded at a very low O2 in N2 are in agreement with ours, which indicated a respiration rate of 0.9 ml CO2/mg/h after 24 h exposure to 1% O2. However, Carlson (1968a) measured less CO2 production than the control in T. confusum adults at 301C in atmospheres of 4.91, 10.15, and 14.90% O2 comparing different % CO2 and % N2 ratios; his CO2
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measurements for these O2 levels were between 1.74 and 1.88 ml CO2/mg/h. These were similar to our results and may be compared at 5%, 10% and 15%, O2 which showed a respiration rate of 2.2–2.6 ml CO2/mg/h (Fig. 6). 4.4. Lethal influence of low O2 levels Many researchers reported that high mortalities under low O2 conditions were obtained only after long exposures, for all development stages (Annis, 1987; Donahaye et al., 1996; Bailey and Banks, 1980; Calderon and Navarro, 1979). Our results showed suppressed respiration at low O2 levels allowing the insects to survive. Calderon and Navarro (1980) reported that the egg stage of T. castaneum was more susceptible than its adult to reduced O2 concentrations and 100% mortality of eggs was obtained only after 96 h when exposed to 2–4% O2 levels. Tunc and Navarro (1983) found that exposure of 0–1 day old T. castaneum eggs to 2–4% O2 at 261C and 20%, 50% and 95% r.h. caused complete mortality after 96 h. Jay and Cuff (1981) reported that upon exposure to B1% O2 at 271C and 60% r.h. for 24 h, mortalities for 16–20 day-old larvae, for 3–6 day-old pupae and for 2–14 day-old adults of T. castaneum were 11.2%, 26% and 99.2%, respectively. Navarro (1978) obtained a high mortality (95%) of T. castaneum adults at 1% O2 only in N2 after 96 h at 261C and 54% r.h. In the present paper, our results showed negligible levels of mortality for adults at low O2 concentrations during the first 24 h of exposure. 4.5. Respiratory quotients RQ values are frequently used as a measurement of insect metabolism. Edwards (1953) stated that the RQ varied according to nutrition type and that RQ values of 1.0, 0.8 and 0.7 accounted for carbohydrate, protein and lipid oxidation respectively. In the present study, the RQ in normal air for young larvae, old larvae, pupae, and adults (Table 2) reflects carbohydrate metabolism for larvae, lipid metabolism for pupae, and carbohydrate–protein metabolism for adults. These types of metabolic substrates were also observed in other insects. RQ values for C. ferrugineus larvae and adults were reported to be in the range of 0.8–0.9 indicating lipid-carbohydrate metabolism during the development of O. surinamensis. White and Sinha (1981) suggested a similar fluctuation in which an RQ of 1.0 was obtained for larvae, 0.76 for pupae and 0.95–1.0 for adults. RQ values measured in normal air for adults were also reported as 1.1 in S. oryzae (Birch, 1947), 1.02 for T. confusum (Carlson, 1966, 1968a) and 0.83 for T. castaneum (Donahaye, 1992). The values which were closer to unity for adults are in agreement with the results reported in this paper at normal atmospheric air (Table 2). At low O2 concentrations, Thunberg (1905) for Tenebrio larvae and Harnisch (1930) for Chironomus larvae reported that the RQ tended to rise owing to increased acidity which drives off CO2, and that the values may even rise above unity. In our study, we also observed a tendency towards a higher RQ as the O2 concentrations decreased (Table 2). Carlson (1968a), however, reported a decrease in RQ for T. confusum adults exposed to low O2 levels. He reported that in atmospheres containing 4.91%, 10.15% and 14.90% O2 in combination with CO2 and N2, RQ values were 0.6, 0.78, and 0.79, respectively. On the other hand, Donahaye (1992) reported that in T. castaneum adults RQ increased above unity during a 24 h exposure to 0.5% O2 in N2 at 261C and 95% r.h. Based on the RQ values before and after exposure, he concluded that aerobic
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metabolism continued even at such a low level of O2. Carlson (1968b), studying the respiratory metabolism of T. confusum adults exposed for 2 h to pure CO2 or N2 at 301C, reported that RQ values were above and below unity, respectively. He suggested anaerobic metabolism in a CO2 atmosphere, and concluded that N2-induced anaerobism caused a less physiological disturbance to insects than CO2. Edwards (1953) suggested that an RQ greater than unity might be explained as an oxidation of organic acids, as an acidosis, as a saturation of fatty acids, or as the conversion of carbohydrate to fat. In larvae under normal atmospheric and food conditions it is convenient to ascribe RQ values greater than unity to conversion of carbohydrate to fat, in order to store up materials for the pupal stage. On the other hand, acidosis might be ascribed to the accumulation of metabolites such as CO2 and lactic acid during anaerobiosis (Edwards, 1953). Keister and Buck (1974) also reported that CO2 production and lactic acid accumulation during anoxia and excess O2 consumption following anoxia might be regarded as an evidence of oxygen debt during the anoxic period. In the present work, a high CO2 production in adults was observed at 3–5% O2. High levels of lactate, alpha-glycerophosphate, glycerol, alanine and malate (Friedlander and Navarro, 1979) were also reported as indicators of an oxygen debt. Similarly, in the present work, increased CO2 production of adults at 3% and 5% O2 might indicate anaerobic metabolism. 4.6. Respiration rate and sealed storage In developing countries, hermetic storage provides a proven solution as an inexpensive and practical method of food storage. It does not require a sophisticated technology which is usually not available to small farmers in developing countries. The most important advantage of this technology is its safety to operators and to consumers. Amongst the other MA options, it is also the only one that does not require equipment such as MA-generators or gas-cylinders (Navarro et al., 1994). In the present work, at O2 levels of 3% and 5%, adults in particular underwent metabolic stress as expressed by their high CO2 production after 24 h exposure. This stress is possibly accompanied by other sublethal effects leading towards mortality as the exposure is extended at the tested O2 levels. This aspect deserves further investigation. Additional studies are needed on the metabolic effects of insects at concentrations of 3–5% O2, which mostly prevail in the sealed storages. These results may help to construct simulation models, and promote the more effective use of the sealed (hermetic) method of storage. Acknowledgements The authors thank Mr. R. Dias for his technical assistance. The first author wishes to express his appreciation to the Ministry of Foreign Affairs of Israel for supporting him during the course of the present work.
References Aliniazee, T.M., 1971. Effect of carbon dioxide gas on respiration of the confused flour beetle. Journal of Economic Entomology 64, 1304–1305.
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Annis, P.C., 1987. Towards rational controlled atmosphere dosage schedules: a review of current knowledge. In: Donahaye, E., Navarro, S. (Eds.), Proceedings of the Fourth International Working Conference of Stored-Product Protection, Tel Aviv, Israel, 1986, pp. 128–148. Annis, P.C., Dowsett, H.A., 1993. Low oxygen disinfestation of grain: exposure periods needed for high mortality. In: Navarro, S., Donahaye, E. (Eds.), Proceedings of the International Conference on Controlled Atmosphere and Fumigation in Grain Storages, Winnipeg, Canada, 1992, pp. 71–83. Bailey, S.W., Banks, H.J., 1975. The use of controlled atmospheres for the storage of grain. In: Proceedings of the First International Working Conference on Stored Product Entomology, Savannah, Georgia, USA, 1974, pp. 362–374. Bailey, S.W., Banks, H.J., 1980. A review of recent studies of the effects of controlled atmospheres on stored product pests. In: Shejbal, J., (Ed.), Controlled Atmosphere Storage of Grains, Proceedings of an International Symposium, Rome, 1980, pp. 101–118. Banks, H.J., Annis, P.C., Henning, R.C., Wilson, A.D., 1980. Experimental and commercial modified atmosphere treatments of stored grain in Australia. In: Shejbal, J., (Ed.), Controlled Atmosphere Storage of Grains, Proceedings of an International Symposium, Rome, 1980, pp. 523–542. Birch, L.C., 1947. The oxygen consumption of the small strain of Calandra oryzae L. and Rhizopertha dominica Fab. as affected by temperature and humidity. Ecology 28, 17–25. Calderon, M., Navarro, S., 1979. Increased toxicity of low oxygen atmospheres supplemented with carbon dioxide on Tribolium castaneum adults. Entomologia Experimentalis et Applicata 25, 39–44. Calderon, M., Navarro, S., 1980. Synergistic effect of CO2 and O2 mixtures on two stored grain insect pests: In: Shejbal, J. (Ed.), Controlled Atmosphere Storage of Grains, Proceedings of an International Symposium, Rome, 1980, pp. 79–84. Calderwood, W.A., 1961. The metabolic rate of the flour beetle, Tribolium confusum. Transactions of the Kansas Academy of Science 64, 150–152. Campbell, A., Sinha, R.N., 1978. Bioenergetics of granivorous beetles, Cryptolestes ferrugineus and Rhyzopertha dominica (Coleoptera: Cucujidae and Bostrichidae). Canadian Journal of Zoology 56, 624–633. Campbell, A., Sinha, R.N., 1990. Analysis and simulation modelling of population dynamics and bioenergetics of Cryptolestes ferrugineus (Coleoptera: Cucujidae) in stored wheat. Researches on Population Ecology 32, 235–254. Carlson, S.D., 1966. Respiration measurement of Tribolium confusum by gas chromatography. Journal of Economic Entomology 59, 1088–1103. Carlson, S.D., 1968a. Respiration of the confused flour beetle in five atmospheres of varying CO2:O2 ratios. Journal of Economic Entomology 61, 94–96. Carlson, S.D., 1968b. Respiration of confused flour beetle in CO2 or N2 and after sublethal fumigation. Journal of Economic Entomology 61, 125–131. Chaudry, H.S., Kapoor, R.P.D., 1967. Studies on the respiratory metabolism of the red flour beetle. Journal of Economic Entomology 60, 1334–1336. Donahaye, E., 1990. Laboratory selection of resistance by the red flour beetle Tribolium castaneum (Herbst) to an atmosphere of low oxygen concentration. Phytoparasitica 18, 189–202. Donahaye, E., 1992. Physiological differences between strains of Tribolium castaneum selected for resistance to hypoxia and hypercarbia, and the unselected strain. Physiological Entomology 17, 219–229. Donahaye, E., Navarro, S., Rindner, M., Azrieli, A., 1996. The combined influence of temperature and modified atmospheres on Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Journal of Stored Products Research 32, 225–232. Dumas, T., Buckland, C.T., Monro, H.A.U., 1969. The respiration of insects at reduced pressures II. The uptake of oxygen by Tenebroides mauritanicus. Entomologia Experimentalis et Applicata 12, 389–402. Edwards, D.K., 1958. Effects of acclimatization and sex on respiration and thermal resistance in Tribolium (Coleoptera: Tenebrionidae). Canadian Journal of Zoology 36, 363–382. Edwards, G.A., 1953. Respiratory metabolism. In: Roeder, K.D. (Ed.), Insect Physiology. Wiley Inc., New York, Chapman & Hall Limited, London, pp. 96–146 (Chapter 5). Friedlander, A., Navarro, S., 1979. The effect of controlled atmospheres on carbohydrate metabolism in the tissue of Ephestia cautella pupae. Insect Biochemistry 9, 79–83.
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Harnisch, O., 1930. Effect of reduced oxygen tension on metabolism in fragments of Chironomus larvae. Zeitschrift fur Vergleichende Physiologie 14, 504–523. Jay, E.G., Cuff, W., 1981. Weight loss and mortality of three life stages of Tribolium castaneum (Herbst.) when exposed to four modified atmospheres. Journal of Stored Products Research 17, 117–124. Keister, M., Buck, J., 1974. Respiration: some exogenous and endogenous effects on rate of respiration. In: Rockstein, M. (Ed.), Physiology of Insecta, Vol. III, 2nd Edition, Academic Press, New York, London, pp. 469–509 (Chapter 7). Kennington, G.S., 1957. Influence of altitude and temperature upon rate of oxygen consumption of Tribolium confusum J. Duval and Camponotus pennsylvanicus modoc Wheeler. Physiological Zoology 30, 305–314. Navarro, S., 1978. The effects of low oxygen tensions on three stored-product insect pests. Phytoparasitica 6, 51–58. Navarro, S., Calderon, M., 1979. Mode of action of low atmospheric pressures on Ephestia cautella (Wlk.) pupae. Separatum Experientia 35, 620. Navarro, S., Donahaye, E., 1972. An apparatus for studying the effects of controlled low pressures and compositions of atmospheric gases on insects. Journal of Stored Products Research 8, 223–226. Navarro, S., Donahaye, E., 1990. Generation and application of modified atmospheres and fumigants for the control of storage insects. In: Champ, B.R., Highley, E., Banks, H.J. (Eds.), Fumigation and Controlled Atmosphere Storage of Grain, Proceedings of an International Conference, ACIAR Proceedings No. 25, Singapore, 1989, pp. 152–165. Navarro, S., Gonen, M., Schwartz, A., 1979. Large scale trials on the use of controlled atmospheres for the control of stored grain insects. In: Proceedings of the Second International Working Conference on Stored-Product Entomology, Ibadan, 1978, pp. 260–270. Navarro, S., Varnava, A., Donahaye, E., 1993. Preservation of grain in hermetically sealed plastic liners with particular reference to storage of barley in Cyprus. In: Navarro, S., Donahaye, E. (Eds.), Proceedings of the International Conference on Controlled Atmosphere and Fumigation in Grain Storages, Winnipeg, Canada, 1992, pp. 223–234. Navarro, S., Donahaye, E., Fishman, S., 1994. The future of hermetic storage of dry grains in tropical and subtropical climates. In: Highley, E., Wright, E.J., Banks, H.J., Champ, B.R. (Eds.), Proceedings of the Sixth International Working Conference on Stored-Product Protection, Vol. 1, Canberra, 1994, pp. 130–138. Park, T., 1936. Studies in population physiology. Journal of Cellular and Comparative Physiology 7, 313–323. Thunberg, D., 1905. Oxygen tension and uptake in Tenebrio larvae. Skandinavisches Archiv fur Physiologie 17, 133–195. Tunc, I., Navarro, S., 1983. Sensitivity of Tribolium castaneum eggs to modified atmospheres. Entomologia Experimentalis et Applicata 34, 221–226. White, N.D.G., Sinha, R.N., 1981. Energy budget for Oryzaephilus surinamensis (Coleoptera: Cucujidae) feeding on rolled oats. Environmental Entomology 10, 320–326.
Respiration of Tribolium castaneum (Herbst) at reduced oxygen concentrations M. Emekcia, S. Navarrob,*, E. Donahayeb, M. Rindnerb, A. Azrielia b
a Plant Protection Department, Faculty of Agriculture, University of Ankara, 06110 Ankara, Turkey Department of Stored Products, The Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel
Accepted 17 August 2001
Abstract Adults, eggs, young and old larvae and pupae of Tribolium castaneum (Herbst) were exposed to atmospheres containing 1%, 2%, 3%, 5%, 10%, and 15% oxygen in nitrogen at 301C and 70% r.h. Respiration rates were determined with a gas chromatograph. The oxygen intake and carbon dioxide output by insects were expressed in ml/insect/h or ml/mg/h. Adults exposed to 21% oxygen required an initial acclimatization period of at least 5 h, after which the respiration rate remained stable. Based on this finding, all the respiration measurements were carried out after an initial adaptation of insects to the respirometer conditions for 24 h. Respiration of eggs, young and old larvae, pupae, and adults at 301C in normal atmospheric air was at rates of 0.0121, 9.25, 8.45, 1.45, and 4.67 ml CO2/insect/h, respectively. Respiration rates of the same stages in terms of insect weight were 0.32, 29.08, 3.33, 0.59 and 2.37 ml CO2/mg insect/h, respectively. At reduced oxygen levels respiration rates of eggs, larvae and pupae were proportional to the oxygen levels. Adult respiration was higher for 3% and 5% oxygen than for normal atmospheric air with rates of 4.77 and 4.98 ml CO2/insect/h, respectively. In adults, RQ values for the same oxygen levels were also higher than for normal atmospheric oxygen and were 1.07 and 1.18, respectively. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Tribolium castaneum; Development stages; Respiration; Reduced oxygen
1. Introduction Modified atmosphere (MA) technology has been widely recognized as a means of preserving stored grain (Bailey and Banks, 1975; Banks et al., 1980; Navarro et al., 1979; Navarro and *Corresponding author. Fax: +972-3-9604428. E-mail address: [email protected] (S. Navarro). 0022-474X/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 4 7 4 X ( 0 1 ) 0 0 0 4 5 - 5
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Donahaye, 1990). Among the MA application methods, the bio-generation of the atmosphere through the respiration of insect pests and microorganisms has been shown to be effective in depleting the oxygen (O2) and increasing the carbon dioxide (CO2) concentration of the intergranular space. Using this process in hermetic storage, it is possible to arrest insect pest development and to minimize storage damage (Navarro et al., 1993). Improvements in gas generation technology can also reduce the cost of MA generation. Such improvements together with the laboratory experiments to demonstrate the effectiveness of low O2 or high CO2 concentrations could lead to a more widespread use of MAs in the future (Annis and Dowsett, 1993). Most of the research on MAs has naturally been concentrated on the effectiveness of different atmospheric gas combinations as a means of insect control. However, the effects of sublethal concentrations of MAs on insects, such as delaying development, impaired metamorphosis and altered fecundity, have not been well documented (Bailey and Banks, 1980). Among the effects of MAs, their influence on insect respiration has also not been sufficiently studied. Knowledge of the respiration rate of insects in storage systems is important, since this information can be utilized in a number of situations such as in calculating its effect on heating of bulk-stored grain. Respiration is also a good index of the physiological responses of the insects to the environment in which they are exposed. Several works have been carried out on the respiratory mechanisms of stored product insect pests, mostly at normal atmospheric air. Park (1936) investigated O2 consumption of Tribolium spp. in relation to body weight, sex, and food medium. Birch (1947) studied the O2 consumption of various developmental stages of S. oryzae (L.), and Rhyzopertha dominica (F.) over a range of temperatures and at different relative humidities (r.h.). They showed that O2 consumption increased as the development proceeded up to the prepupal stage. Calderwood (1961) conducted studies on the O2 consumption of Tribolium confusum du Val. and Tribolium castaneum in relation to the temperatures between 151C and 371C, and found a positive correlation between metabolic rate and temperature. Chaudry and Kapoor (1967) investigated the respiratory metabolism of T. castaneum in relation to temperature. Carlson (1966, 1968a, b) measured the respiration of T. confusum under different CO2:O2 ratios and reported that sublethal exposure to CO2 or N2 reduced the O2 consumption in adults. Dumas et al. (1969) investigated the larval respiration of Tenebroides mauritanicus (L.). They reported that both O2 consumption and CO2 production were progressively reduced as the pressure was lowered from 200 to 35 mmHg. Aliniazee (1971) showed that high CO2 levels (8–16%) severely suppressed respiration in T. confusum. Navarro and Calderon (1979) investigated the effects of low atmospheric pressures and low O2 concentrations on pupal respiration, mortality, and weight loss in Ephestia cautella (Walker), and stated that respiration was suppressed in both types of atmospheres at an O2 concentration equivalent to 3%. Campbell and Sinha (1978), studying the bio-energetics of Cryptolestes ferrugineus (Stephens), stated that prepupae, pupae, and adults had a lower respiration rate than did the developing larvae. White and Sinha (1981), and Campbell and Sinha (1990) reached the same conclusion in Oryzaephilus surinamensis (L.) and C. ferrugineus, respectively. Donahaye (1992) investigated the metabolic rate in terms of O2 uptake, CO2 production, and losses in wet and dry weights in adults of T. castaneum which were exposed to two kinds of MAs. He found that respiration was higher in the unselected strain as compared with both the high carbon dioxide concentration (HCC), and low oxygen concentration (LOC) selected strains at 261C and 95% r.h. Many laboratory studies
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have concentrated on the effectiveness of atmospheres containing 0–1% O2 particularly on the control of the adult stage of insects, whereas, atmospheres of 1–5% O2 concentrations are easier to obtain and maintain economically in sealed storages for the application of MAs (Annis and Dowsett, 1993). There is a lack of information on the effect of low O2 levels, particularly those higher than 1%, on the different developmental stages of stored grain insects in terms of both lethal and sublethal aspects. Such information can be particularly useful when considering simulation models. The objective of this work was to study the respiration rates of the development stages of T. castaneum at low levels of O2 which prevail when atmospheres under gas tight conditions are modified by the metabolic activity of insects.
2. Materials and methods 2.1. Insect culture techniques T. castaneum eggs were obtained from 1000 to 2000 young adults placed in 1-l jars that contained approximately 250 g of flour and 5% yeast (by weight). Every three days the jars were sieved for egg removal. These adults were used for oviposition for up to one month. Approximately 2000–3000 three day old eggs were placed in jars, containing B500 g finely ground wheat and 5% yeast. The medium was later sieved to obtain young larvae, mature larvae, pupae and adults (Donahaye, 1990). 2.2. Modified atmospheres The respiration rate of T. castaneum was measured in atmospheres containing 1%, 2%, 3%, 5%, 10% or 15% O2 in N2 at 301C. Normal air served as a control. Gas mixtures were prepared as described by Donahaye (1990), and maintained at a flow rate of 15 ml/min at 70% r.h. (Navarro and Donahaye, 1972). Table 1 shows the different development stages, the number of individuals and age groups of T. castaneum used in studying respiration rates at low oxygen concentrations. Table 1 Numbers and the ages of Tribolium castaneum at the stages used for testing their respiration rates Development stage
Number of insects per treatment
Age of insectsa (days)
Eggs Young larvae Old larvae Pupae Adults
300 100 100 100 100
0–1 7–10 18–22 0–1 10–14
a Age for larval stages from egg stage; pupae from pupation; and adults from emergence.
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2.3. Exposure flasks The flasks consisted of 50–60 ml bottles with 2 cm wide necks. Their volumes were measured carefully before the experiments. The bottles were equipped with silicon septa and aluminum lids. A crimper was used to seal the bottles tightly. Two N.14 syringe needles of 0.65 32 mm2 were inserted through the septa. One served as an inlet for the gas mixture while the other served as an outlet.
2.4. Exposure techniques Known numbers of insects (Table 1) were weighed and transferred to the B60 ml experimental bottles containing 200 mg flour for all development stages, except eggs and pupae, which did not need the food. After closing, the bottles were connected to the exposure apparatus. A soap-bubble flow meter was attached to the outlet needle of each bottle to verify the flow rate through the bottle and to ensure that there were no blockages in the system. The bottles were held at 301C in a thermostatically controlled room. Following 24 h of exposure (for all stages except eggs), the air composition within the bottle was measured by sampling with a 1 ml ‘‘pressure-lock’’ syringe through the silicon septum. The inlet and outlet ports of the bottles were then blocked tightly for approximately 1 h (24 h for eggs) by means of metal clamps. At the end of this period flasks were sampled as described previously. Gas concentrations were determined using a Tracer 565 gas chromatograph equipped with twin thermal conductivity cells, dual columns (each 40 1/1600 i d), and a sample loop thermostatically regulated at 501C. Column 1 was packed with ‘‘Porapak Q’’, while column 2 was packed with ‘‘molecular sieve 5a’’. Percentage concentration of the gas components was computed by a Spectra Physics SP 4100 integrator. Preliminary experiments at a normal atmospheric air pressure showed that adults had to be exposed for acclimatization for not o5 h prior to experimentation (Fig. 1). All stages except eggs were, therefore, exposed for a 24 h acclimatization period before gas sampling.
Carbon dioxide production
6.0
5.0
µl CO 2 /insect/h
4.0
3.0 µl CO 2 /mg/h 2.0
1.0 0
8
16
24
32
40
48
Time (h)
Fig. 1. Change in respiration rate of Tribolium castaneum adults during acclimatization in normal atmospheric air at 301C and 70% r.h.
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The possibility of leakage due to successive sampling from the small volume of experimental flasks (50–60 ml) with a relatively large sample sizes (1 ml) was investigated. Although we did not find any statistical difference between the first two successive samples taken from the same flasks which were in the gas tight position, the method was not suitable for the detection of the tiny O2 consumption of eggs exposed to low oxygen concentrations. It was suspected that slight leakage occurred. In order to avoid the effects of possible leakages, results for eggs were expressed on the basis of CO2 production instead of O2 uptake. Since one of the aims of this study was to provide more information on respiration we preferred to give the CO2 production per mg body weight per h together with the CO2 production per insect per h in each figure for the different developmental stages. Such information is generally lacking in the literature.
3. Results Results showed that the respiration rate of immature stages increased with the increase of O2 levels. The most pronounced suppressant effect of low O2 concentrations on respiration was observed with eggs (Fig. 2) and young larvae (Fig. 3). For these developmental stages, CO2 production was lower at concentrations of up to 3% O2 as compared to the controls. At concentrations of 1–15% O2, respiration of old larvae appeared higher than that of young larvae (Fig. 4). However, examination of the respiration rate by body weight reveals that young larvae respired much more rapidly than old larvae (Figs. 3 and 4). A further increase of O2 level to 21% leads to even higher respiration in young larvae. As development proceeded, respiration was markedly suppressed when O2 was lowered to 1% in old larvae and to some extent in pupae (Figs. 4 and 5). RQ values in normal atmospheric air were 1.02 and 0.72 for old larvae and pupae, respectively (Table 2.). Unlike prepupal stages, in adults high CO2 production was obtained at 3–10% O2 (Fig. 6); but adult respiration was less affected by the experimental range of O2 concentrations than that of younger larvae or eggs. The RQ value in normal air for adults was 0.89 (Table 2).
Carbon dioxide production
0.35 0.30 0.25
µl CO 2 /mg/h
0.20
µl CO 2 /10 eggs/h
0.15 0.10 0.05 0.00 0
5
10 15 Oxygen concentration (%)
20
Fig. 2. Respiration rates of Tribolium castaneum eggs in low oxygen concentrations in nitrogen at 301C and 70% r.h.
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Carbon dioxide production
28 24 µl CO 2 /mg/h
20 16
µl CO2 /insect/h
12 8 4
0
5
10 15 Oxygen concentration (%)
20
Fig. 3. Respiration rates of Tribolium castaneum young larvae in low oxygen concentrations in nitrogen at 301C and 70% r.h. 9 Carbon dioxide production
8 7
µl CO2 /insect/h
6 5
µl CO2 /mg/h
4 3 2 1 0 0
5
10 15 Oxygen concentration (%)
20
Fig. 4. Respiration rates of Tribolium castaneum old larvae in low oxygen concentrations in nitrogen at 301C and 70% r.h.
With some exceptions RQ values generally tended to increase at all life stages as the O2 concentrations decreased (Table 2).
4. Discussion 4.1. Adult respiration in air Respiration of Tribolium spp. has mostly been studied with the adult stage and at normal atmospheric air pressure. A comparison of our results for oxygen intake of adults with data from the literature is shown in Table 3 although differences in temperature, r.h. and particularly
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Carbon dioxide production
1.50
1.25 µl CO 2 /insect/h 1.00 µl CO 2 /mg/h
0.75 0.50 0.25 0
5
10 15 Oxygen concentration (%)
20
Fig. 5. Respiration rates of Tribolium castaneum pupae in low oxygen concentrations in nitrogen at 301C and 70% r.h.
Table 2 Respiratory quotients of life stages of Tribolium castaneum at 301C and 70% r.h. at various concentrations of oxygen in nitrogen O2 concentration (%)
1 2 3 5 10 15 21
Life stages Eggs
Young larvae
Old larvae
Pupae
Adults
F F F 1.85 0.96 0.92 0.83
2.40 1.05 0.95 1.04 1.00 1.00 1.01
0.93 1.12 1.06 1.03 1.00 1.06 1.02
1.07 1.16 0.98 1.18 0.78 0.64 0.72
1.22 0.92 1.07 1.18 1.03 0.96 0.88
6.0
Carbon dioxide production
5.5
µl CO 2 /insect/h
5.0 4.5 4.0 3.5 µl CO 2 /mg/h
3.0 2.5 2.0 1.5 1.0 0.5 0
5
10 15 Oxygen concentration (%)
20
Fig. 6. Respiration rates of Tribolium castaneum adults in low oxygen concentrations in nitrogen at 301C and 70% r.h.
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Table 3 Comparative data on respiration rates of Tribolium spp. adults in air from the literature Species
Temperature (1C)
Respiration ratesa ml O2/insect/h
T. confusum ‘‘ ‘‘ ‘‘ ‘‘ ‘‘ ‘‘
29 25 37 30 30
T. castaneum ‘‘ ‘‘ ‘‘ ‘‘ ‘‘ ‘‘ ‘‘
30 35 26.6 26 20 25 30 35
a
ml O2/mg/h
Source ml CO2/mg/h
1.79–2.31 1.6 1.6
2.64 2.69
Park (1936) Edwards (1935) Kennington (1957) Calderwood (1961) Calderwood (1961) Carlson (1966) Carlson (1968a)
4.26 2.15 1.19 1.68 2.37 3.31
Chaudry and Kapoor (1967) Chaudry and Kapoor (1967) Aliniazee (1971) Donahaye (1992) Present work Present work Present work Present work
3.14 5.83
1.09 2.39
2.49 3.71 5.73 7.15
1.36 1.83 2.97 3.55
Converted to ml wherever necessary.
acclimatization periods need to be taken into consideration. Park (1936) reported the O2 consumption of T. confusum in the range of 1.79–2.31 ml O2/mg/h at 281C. His studies were detailed and also included sexual differences and conditioning of food. According to Edwards (1958) T. confusum respired 1.6 ml O2/mg/h (converted from mm3/mg/h) at 261C. Similar results were obtained by Kennington (1957) at 291C. Chaudry and Kapoor (1967) obtained a lower O2 uptake by T. castaneum of 1.09 ml O2/mg/h at 301C, increasing to 2.39 ml O2/mg/h at 351C. Our results showing a respiration rate of 1.83 ml O2/mg/h at 251C and 2.97 ml O2/mg/h at 301C are in agreement with the data in the literature. Calderwood (1961) reported the O2 uptake by T. confusum adults at 251C and 371C as 3.14 ml O2/insect/h (converted from mmol/h) and 5.83 ml O2//insect/h (converted from mmol/h), respectively. In our experiment, O2 uptake was found to be 5.73 ml O2/insect/h at 301C. Aliniazee (1971) reported that CO2 production in T. castaneum at 26.61C was 4.26 ml CO2/mg/h (converted from mg/g/h). Donahaye (1992) stated that in T. castaneum, CO2 expiration was 2.15 ml CO2/mg/h (converted from mmol/mg/min) at 261C and 95% r.h. In two separate works, Carlson (1966, 1968a) reported that CO2 production in T. confusum at 301C was 2.64 ml CO2/mg/h (converted from mg/mg/min) and 2.69 ml CO2/mg/h (converted from mmol/mg/min), respectively. In these studies, the acclimatization period was reported as a maximum of 1 h (Carlson, 1966, 1968a). In the present work, we found that before acclimatization respiration rate was 2.9 ml CO2/ mg/h and after 5 h of acclimatizing insects to the experimental conditions the respiration rate was 2.0 ml CO2/mg/h (Fig. 1). The difference between the results of these studies and ours can partly be attributed to the differences in acclimatization of insects to the test conditions.
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4.2. Respiration of different developmental stages of other species at normal atmospheric pressure Birch (1947), who studied the respiration of S. oryzae at 301C and 70% r.h. at normal atmospheric air concluded that the rate of oxygen consumption per insect increased during the development from the egg to prepupal stage, then fell during pupation. In the same work, O2 consumption in S. oryzae populations composed of young larvae, old larvae, mainly pupae (84%) and 1-week-old adults was given as 5.30, 26, 5.77, and 5.40 ml/insect/h (converted from mm3/insect/h), respectively. In our experiment, we observed a similar variation in respiration rates according to life stages, but respiration was higher in young larvae than in older ones in normal air (Figs. 3 and 4). In this respect, our results were closer to those of Campbell and Sinha (1978). They stated a reduction in O2 uptake of C. ferrugineus on the basis of dry body weight from 149 ml O2/mg/h in first instar larvae to 27.5 ml O2/mg/h in the adult stage in normal atmospheric air at 301C and 70% r.h. White and Sinha (1981) reported the same respiration pattern in O. surinamensis. They found that the hourly O2 consumption per insect at 301C and 80% r.h. increased to a maximum of 108 ml, when the majority of insects were entering the second instar, decreasing to 0.4 ml at pupation and rising to 4.4 ml during the first few days of the adult stage. 4.3. Respiration at reduced O2 levels We had difficulties in comparing our findings to those in the literature due to lack of information on the respiration of T. castaneum at reduced O2 concentrations, particularly on the variations in response to different development stages. Our results showed that respiration tended to increase as the O2 concentration increased from 1% to 21% for all development stages except the adult. During the course of this work, the respiration rate was suppressed at O2 levels lower than 5% in eggs and young larvae (Figs. 2 and 3). Low O2 levels were more effective in reducing the respiration rate of young larvae than old larvae. On the other hand the CO2 production expressed per insect was higher in young larvae than in old larvae in the control (21% O2) (Figs. 3 and 4). Pupal respiration decreased as O2 concentrations dropped (Fig. 5). Similar results on respiration of E. cautella pupae were reported at reduced O2 atmospheres at 261C and 93–99% r.h. (Navarro and Calderon, 1979). Accordingly CO2 production by E. cautella was approximately 0.1, 0.2 and 0.35 ml CO2/100 mg/h at O2 concentrations of 1%, 3% and 21%, respectively. These figures were in agreement with our findings which showed relatively less disruption of the pupal stage at low O2 concentrations. One of the most interesting results in this study was the increased respiration rate of adults at 3% and 5% O2 (Fig. 6) which can be considered as a compensation response to the O2 deficiency. Donahaye (1992) provided data on the physiological differences between unselected and selected strains of adult T. castaneum for hypoxia and hypercarbia at 261C and 95% r.h. We calculated that CO2 production was reduced from 1.88 to 1.34 ml CO2/mg/h (converted from mmol/mg/min) for the unselected strain when respiration was measured initially and after exposure for 24 h to 0.5% O2 in N2. His results, though recorded at a very low O2 in N2 are in agreement with ours, which indicated a respiration rate of 0.9 ml CO2/mg/h after 24 h exposure to 1% O2. However, Carlson (1968a) measured less CO2 production than the control in T. confusum adults at 301C in atmospheres of 4.91, 10.15, and 14.90% O2 comparing different % CO2 and % N2 ratios; his CO2
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measurements for these O2 levels were between 1.74 and 1.88 ml CO2/mg/h. These were similar to our results and may be compared at 5%, 10% and 15%, O2 which showed a respiration rate of 2.2–2.6 ml CO2/mg/h (Fig. 6). 4.4. Lethal influence of low O2 levels Many researchers reported that high mortalities under low O2 conditions were obtained only after long exposures, for all development stages (Annis, 1987; Donahaye et al., 1996; Bailey and Banks, 1980; Calderon and Navarro, 1979). Our results showed suppressed respiration at low O2 levels allowing the insects to survive. Calderon and Navarro (1980) reported that the egg stage of T. castaneum was more susceptible than its adult to reduced O2 concentrations and 100% mortality of eggs was obtained only after 96 h when exposed to 2–4% O2 levels. Tunc and Navarro (1983) found that exposure of 0–1 day old T. castaneum eggs to 2–4% O2 at 261C and 20%, 50% and 95% r.h. caused complete mortality after 96 h. Jay and Cuff (1981) reported that upon exposure to B1% O2 at 271C and 60% r.h. for 24 h, mortalities for 16–20 day-old larvae, for 3–6 day-old pupae and for 2–14 day-old adults of T. castaneum were 11.2%, 26% and 99.2%, respectively. Navarro (1978) obtained a high mortality (95%) of T. castaneum adults at 1% O2 only in N2 after 96 h at 261C and 54% r.h. In the present paper, our results showed negligible levels of mortality for adults at low O2 concentrations during the first 24 h of exposure. 4.5. Respiratory quotients RQ values are frequently used as a measurement of insect metabolism. Edwards (1953) stated that the RQ varied according to nutrition type and that RQ values of 1.0, 0.8 and 0.7 accounted for carbohydrate, protein and lipid oxidation respectively. In the present study, the RQ in normal air for young larvae, old larvae, pupae, and adults (Table 2) reflects carbohydrate metabolism for larvae, lipid metabolism for pupae, and carbohydrate–protein metabolism for adults. These types of metabolic substrates were also observed in other insects. RQ values for C. ferrugineus larvae and adults were reported to be in the range of 0.8–0.9 indicating lipid-carbohydrate metabolism during the development of O. surinamensis. White and Sinha (1981) suggested a similar fluctuation in which an RQ of 1.0 was obtained for larvae, 0.76 for pupae and 0.95–1.0 for adults. RQ values measured in normal air for adults were also reported as 1.1 in S. oryzae (Birch, 1947), 1.02 for T. confusum (Carlson, 1966, 1968a) and 0.83 for T. castaneum (Donahaye, 1992). The values which were closer to unity for adults are in agreement with the results reported in this paper at normal atmospheric air (Table 2). At low O2 concentrations, Thunberg (1905) for Tenebrio larvae and Harnisch (1930) for Chironomus larvae reported that the RQ tended to rise owing to increased acidity which drives off CO2, and that the values may even rise above unity. In our study, we also observed a tendency towards a higher RQ as the O2 concentrations decreased (Table 2). Carlson (1968a), however, reported a decrease in RQ for T. confusum adults exposed to low O2 levels. He reported that in atmospheres containing 4.91%, 10.15% and 14.90% O2 in combination with CO2 and N2, RQ values were 0.6, 0.78, and 0.79, respectively. On the other hand, Donahaye (1992) reported that in T. castaneum adults RQ increased above unity during a 24 h exposure to 0.5% O2 in N2 at 261C and 95% r.h. Based on the RQ values before and after exposure, he concluded that aerobic
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metabolism continued even at such a low level of O2. Carlson (1968b), studying the respiratory metabolism of T. confusum adults exposed for 2 h to pure CO2 or N2 at 301C, reported that RQ values were above and below unity, respectively. He suggested anaerobic metabolism in a CO2 atmosphere, and concluded that N2-induced anaerobism caused a less physiological disturbance to insects than CO2. Edwards (1953) suggested that an RQ greater than unity might be explained as an oxidation of organic acids, as an acidosis, as a saturation of fatty acids, or as the conversion of carbohydrate to fat. In larvae under normal atmospheric and food conditions it is convenient to ascribe RQ values greater than unity to conversion of carbohydrate to fat, in order to store up materials for the pupal stage. On the other hand, acidosis might be ascribed to the accumulation of metabolites such as CO2 and lactic acid during anaerobiosis (Edwards, 1953). Keister and Buck (1974) also reported that CO2 production and lactic acid accumulation during anoxia and excess O2 consumption following anoxia might be regarded as an evidence of oxygen debt during the anoxic period. In the present work, a high CO2 production in adults was observed at 3–5% O2. High levels of lactate, alpha-glycerophosphate, glycerol, alanine and malate (Friedlander and Navarro, 1979) were also reported as indicators of an oxygen debt. Similarly, in the present work, increased CO2 production of adults at 3% and 5% O2 might indicate anaerobic metabolism. 4.6. Respiration rate and sealed storage In developing countries, hermetic storage provides a proven solution as an inexpensive and practical method of food storage. It does not require a sophisticated technology which is usually not available to small farmers in developing countries. The most important advantage of this technology is its safety to operators and to consumers. Amongst the other MA options, it is also the only one that does not require equipment such as MA-generators or gas-cylinders (Navarro et al., 1994). In the present work, at O2 levels of 3% and 5%, adults in particular underwent metabolic stress as expressed by their high CO2 production after 24 h exposure. This stress is possibly accompanied by other sublethal effects leading towards mortality as the exposure is extended at the tested O2 levels. This aspect deserves further investigation. Additional studies are needed on the metabolic effects of insects at concentrations of 3–5% O2, which mostly prevail in the sealed storages. These results may help to construct simulation models, and promote the more effective use of the sealed (hermetic) method of storage. Acknowledgements The authors thank Mr. R. Dias for his technical assistance. The first author wishes to express his appreciation to the Ministry of Foreign Affairs of Israel for supporting him during the course of the present work.
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