midity is required, two selenoid valves turn on simultaneously; each allowing compressed air to
flow through two nozzles. Air entrance to the chamber had a velocity of 5 ms and air velocity
inside the chamber averaged 2.5 – 3.5 ms depend- ing on location. Length of treatment was mea-
sured from the time of sealing the chamber and initiating the treatment.
2
.
3
. Statistical analysis At least three replicates were used and at least
240 eggs or 240 larvae were used in each replicate. A total of 45 872 eggs and 38 248 larvae were
used in the experiment. Data are presented as percentage of mortality, which were corrected for
natural and handling mortality Abbott, 1925. Data were analyzed using ANOVA, and means
separation was conducted using the Tukey test. LT
50
, LT
99
and LT
99.9968
lethal temperatures at which 50, 99 and 99.9968 mortality occurs, and
fiducial limits were estimated according to Finney 1971 using SAS 1996 for eggs of A. obliqua
L. exposed to 0 kPa O
2
+ 50 kPa CO
2
at 51, 54 and 55°C for 240 min.
3. Results and discussion
A typical profile of the changes in the tempera- ture of the chamber air supply and return air,
and the concentration of O
2
and CO
2
recorded during a treatment of 0 kPa O
2
+ 50 kPa CO
2
at 48°C for 220 min is shown in Fig. 1. Temperature
and CO
2
increased rapidly, while O
2
concentra- tion decreased rapidly in the chamber. Depending
on the temperaturetime program and atmosphere used, the desired temperature of the supplied air is
usually reached in 20 min, the desired return air temperature in 20 – 25 min, the target O
2
concentration in 15 – 20 min, and the target CO
2
concentration in 30 – 35 min. The mortality in air at 44°C for 160 min was
very low Table 1. Corrected mortality was 4.7, 47.2, 24.7 and 8.4 in eggs and larvae of A.
obliqua and A. ludens, respectively. Average mor- tality increased very significantly when insects
were exposed to CA at 44°C for 160 min. Gener- ally, mortality was higher with the use of low O
2
0 kPa than with the use of high CO
2
concentra- tion 20 kPa. The low O
2
0 kPa and high CO
2
concentrations 50 kPa caused a synergistic effect compared with the separate use of these two
gases. On the basis of that we decided to use this combined CA atmosphere in all later experiments.
In previous preliminary studies Yahia et al., 1997 using fewer insects, we have found that
44°C for 160 min in air or in CA caused 100 mortality. The variations observed with these
studies might be due to several factors such as the different rearing conditions of the insects, temper-
ature and conditions of transport. Insects used in preliminary studies were obtained from a different
source.
Mortality was very high in air at 48°C for 220 min, and slightly increased in CA Table 1. A
100 mortality of larvae of A. ludens and A. obliqua was achieved in air or in CA at 48°C for
220 min. Eggs were more tolerant than larvae, and reached only an average mortality of 79.6 to
97.7 in air and CA at 48°C for 220 min. A 100 mortality was achieved in eggs of both species in
air at 52°C for 240 min. However, CA at 52°C for 240 min caused 100 mortality of all except the
Fig. 1. A profile of the changes in the temperature of supply and return air, and the O
2
and CO
2
concentrations inside the chamber during a treatment consisting of 0 kPa O
2
+ 50 CO
2
for 220 min.
Table 1 Corrected mortality of eggs and third instar larvae of A. ludens and A. oblique at different atmospheres kPa O
2
–kPa CO
2
, temperatures °C and exposure times min
a
Temperature Atmosphere
Time min A. ludens
A. obliqua °C
kPa O
2
–kPa CO
2
Eggs Larvae
Eggs Larvae
160 4.67
Air 47.24 9 14.1
44 24.67 9 15.0
8.35 9 0.4 160
– 0–0
52.93 9 5.61 44
50.81 9 20.8 83.89 9 2.0
160 37.58 9 1.1
26.84 9 2.18 44
55.92 9 6.9 13–20
34.89 9 1.0 0–50
160 44
– 73.03 9 9.04
98.76 9 0.2 59.46 9 5.7
80 19.53 9 11.9
61.77 9 17.0 44
7.49 9 3.6 0–50
58.09 9 15.6 240
55.10 9 22.5 95.93 9 4.5
0–50 85.03 9 15.0
44 99.18 9 0.5
220 71.67 9 23.8
100.00 9 0.0 48
78.93 9 21.1 Air
100.00 9 0.0 220
79.59 9 15.6 100.00 9 0.0
0–50 97.74 9 2.3
48 100.00 9 0.0
80 34.30 9 14.4
100.00 9 0.0 48
40.47 9 13.0 0–50
85.83 9 1.9 160
100.00 9 0.0 98.60 9 0.3
0–50 32.39 9 35.6
48 99.86 9 0.1
240 76.10 9 20.3
69.13 9 6.1 48
69.29 9 22.2 0–50
78.44 9 4.2 51
Air 240
85.36 9 1.5 100.00 9 0.0
100.00 9 0.0 100.00 9 0.0
240 62.49 9 10.3
100.00 9 0.0 51
83.84 9 16.2 0–50
100.00 9 0.0 240
100.00 9 0.0 100.00 9 0.0
Air 100.00 9 0.0
52 100.00 9 0.0
240 63.35 9 36.6
100.00 9 0.0 52
100.00 9 0.0 0–50
100.00 9 0.0 240
100.00 9 0.0 100.00 9 0.0
Air 100.00 9 0.0
54 100.00 9 0.0
240 83.01 9 17
100.00 9 0.0 54
100.00 9 0.0 0–50
100.00 9 0.0 240
100.00 9 0.0 100.00 9 0.0
Air 100.00 9 0.0
55 100.00 9 0.0
80 100.00 9 0.0
100.00 9 0.0 55
100.00 9 0.0 0–50
100.00 9 0.0 160
92.00 9 8.0 0–50
100.00 9 0.0 55
86.10 9 13.9 100.00 9 0.0
240 100.00 9 0.0
100.00 9 0.0 55
100.00 9 0.0 0–50
100.00 9 0.0
a
Values in parenthesis indicate standard error of the mean
eggs of A. obliqua, which had an average mortal- ity of 63.4. The mortality at 54°C was somewhat
similar to that achieved at 52°C, except that there was a slight increase in the mortality of eggs of A.
obliqua in CA 83.0 instead of 63.4 at 52°C. Air and CA at 55°C caused 100 mortality in both
stages of the 2 species, except in eggs in CA for 160 min. It is possible that the anaesthetic effect
of CO
2
on the insect is responsible for increasing their tolerance to high temperature and decreasing
their mortality, as it was the case during the exposure of eggs of A. obliqua at 52 and 54°C.
At 44°C, mortality increased as the duration time increased from 80 to 160 to 240 min Table
1. At 48°C mortality also increased when the duration time increased from 80 to 160 min
Table 1. However, mortality was similar for eggs of A. obliqua and eggs and larvae of A. ludens,
and slightly lower in larvae of A. obliqua after 240 min compared with 160 min. At 55°C mortality
was more uniform and statistically similar at 80, 160 and 240 min Table 1. Therefore, the increase
in the duration time of treatment is effective only at temperatures 5 48°C, but has no effect at
55°C. These results indicate that increasing the temperature is more effective for insect mortality
than increasing the duration time.
The mean estimated temperatures for LT
50
s, LT
99
s, and LT
99.9968
s of eggs of A. obliqua the most tolerant stage of both insects exposed to 0
kPa O
2
+ 50 kPa CO
2
for 240 min were 49.4°C with a range of 49.02 – 49.75, 54.8°C with a
range of 54.43 – 55.26, and 60.9°C, respectively Table 2.
Therefore, high temperature, especially in CA is efficient in achieving insect mortality. Vapor heat
treatment at \ 95 RH to a core temperature of 47°C which was held for 15 min was also shown
to meet requirements for a quarantine disinfesta- tion treatment of ‘Kensington’ mangoes against
Queensland fruit fly Bactrocera tryoni and the Mediterranean fruit fly Ceratitis capitata Wiede-
mann Heather et al., 1997. The addition of CA to heat treatments was shown to reduce the time
to achieve the same levels of insect mortality Whiting et al., 1991; Carpenter and Potter, 1994;
Neven and Mitcham, 1996. However, in our work we have seen variable results. For example,
CA significantly increased the mortality over that in air at 44°C but only very slightly at 48°C. At
51, 52 and 54°C, CA caused less mortality of eggs of A. obliqua than in air, although not statistically
different. The toxicity of CA for insects, especially the higher concentration of CO
2
has been known to entomologists for a long time Benschoter et
al., 1981. However, insect response to anoxia and hypoxia is not well understood Fleurat-Lessard,
1990; Yahia, 1998. Some species of insects in- cluding fruit flies, at some of their life stages, live
in an environment with higher concentration of CO
2
andor lower concentration of O
2
, as it is the case during the development of some stages of the
insect inside the fruit. Because CO
2
hydrate is carbonic acid, it must produce acidification and
act indirectly on the membranes by modifying their permeability Nicolas and Sillans, 1989.
Mixtures of CO
2
and air may be more rapidly lethal to some species than pure CO
2
Baily and Banks, 1975, 1980. Lethal effects of CO
2
were shown to be not clearly proportional to concen-
tration, suggesting that there may be no advan- tage
to increasing
gas concentration
above threshold level Benschoter et al., 1981. A com-
plete insect mortality by CO
2
seems necessary since the off-spring of the survivors may show
increased resistance, which would necessitate longer exposure periods to the same concentration
of CO
2
to obtain similar mortality Bond and Buckland, 1979; Navarro et al., 1985.
Soderstrom et al. 1990 have found that low O
2
0.5 kPa is less effective than high concentration of CO
2
in the mortality of codling moth at 25°C. However, Shellie et al. 1997 using very few
insects found that reduced O
2
concentration 1 kPa was more lethal to A. ludens larvae than was
an enriched CO
2
atmosphere 20 kPa, which is in agreement with what we are reporting here on A.
ludens and A. obliqua. The larval mortality of both species was higher in low O
2
than in high CO
2
concentration Table 1. Our results indicated that eggs of A. ludens and
A. obliqua were more tolerant than third instar larvae Table 1. This was also reported for
Queensland fruit fly B. tryoni and the Mediter- ranean fruit fly C. capitata by Heather et al.,
1997. The higher sensitivity of larvae to CA and heat compared to eggs is probably due to their
higher metabolic activity, and to their increased area which permits more gas diffusion. Eggs and
larvae of fruit flies have been shown to increase their tolerance to heat with age up to certain
stages Jang, 1991; Corcoran, 1993. Soderstrom et al. 1990 reported that diapausing larvae of the
codling moth Lepidoptera:Tortricidae is the most tolerant stage to O
2
deficient and CO
2
en- riched atmospheres. Fifth instar larvae was re-
ported to be the most tolerant life stage of Epiphyas post6ittana to 0.4 kPa O
2
with 5 kPa CO
2
at 40°C Whiting et al., 1991. Our results also indicated that eggs of A. obliqua are slightly
more tolerant to heat and to CA than eggs of A. ludens. Cydia pomonella eggs were equally suscep-
tible to CA with O
2
concentration of 0.5 – 2 kPa Soderstrom et al., 1991, but concentrations \ 2
kPa were less effective Soderstrom et al., 1991; Whiting et al., 1996.
Table 2 Average observed mortality of eggs and probit analysis of
A. obliqua in 0 kPa O
2
+ 50 kPa CO
2
at 51, 54 and 55°C for 240 min
a
Dead insects Total insects
Temperature Mortality
°C 816
617 51
75.6 54
621 96.5
859 782
100 782
55 n = 3
LT
50
= 49.43°C
49.02–49.75 m 9 S.E. = 0.433 9 0.0277
LT
99
= 54.80°C
54.43–55.26 x
2
= 243.789
LT
99.9968
= 60.97°C
Covariance = −0.09877
a
n, number of treatments; m 9 S.E., slope 9 standard error; x
2
, chi square; LT, lethal temperature. Numbers in parenthesis are the fiducial limits, at 95 confidence level.
Our results showed that temperature of 55°C resulted in a homogeneous mortality regardless of
the atmosphere used. Whiting et al. 1996 re- ported that temperatures \ 40°C resulted in a
short, homogeneous response for all leafroller species regardless of the O
2
or CO
2
atmosphere composition.
4. Conclusions
