beneficial for preserving the postharvest quality of several fruits Yahia, 1998. The lethal MACA
effects on insects and beneficial effects on com- modity is stimulating research interest to develop
these treatments as quarantine systems for differ- ent insects in different crops. Some mango culti-
vars such as ‘Keitt’ are tolerant to insecticidal extreme atmospheres 0.1 – 0.5 kPa O
2
andor 50 – 80 kPa CO
2
for up to 5 days at 20°C Yahia, 1993, 1994, 1998; Yahia and Tiznado-Hernandez,
1993; Yahia and Vazquez-Moreno, 1993; Yahia and Paull, 1997; Yahia et al., 1989. Several in-
sects can be killed by heat andor by insecticidal atmospheres Armstrong, 1994; Hallman, 1994.
The time required for mortality varies with species and is affected by the life stage, the climatic
conditions temperature, relative humidity, and the atmosphere composition. Mortality is usually
higher at higher temperatures Armstrong, 1994. Short periods of elevated temperatures in combi-
nation with CA are effective in controlling insect pests Yahia, 1998; Yahia and Paull, 1997; Yahia
et al., 1997. Insect mortality by CA and MA is faster at higher temperatures Hallman, 1994.
Whiting and Van Den Heuvel 1995 have shown that CA at 40°C significantly reduced the dura-
tion of exposure required for 100 mortality of Tetranychus urticae Koch. Carbon dioxide at 50
kPa for 4 h and 100 kPa for 7 h at 23°C caused only 6.0 and 6.9 mortality of the Caribbean
fruit fly A. suspensa Loew larvae, respectively, and 100 kPa N
2
killed 7.4 of larvae Benschoter et al., 1981. Sixty hours of exposure to 100 kPa
N
2
were required to kill all the larvae. From a practical
standpoint, a
quarantine treatment
should be accomplished in a shorter period of time. Mango fruit is fairly tolerant to heat Yahia
et al., 1997, and thus quarantine treatments have been developed based on the use of hot water or
hot air Heather et al., 1997. Soderstrom et al. 1991 have shown that CA is more effective for
the control of Cydia pomonella L. when com- bined with high temperatures. Whiting et al.
1991 have shown that lower concentration of O
2
1 kPa at high temperatures increased the mortal- ity of several insects. The use of high levels of
CO
2
60 – 90 kPa and low levels of O
2
1 kPa decreased the mortality time of Tribolium casta-
neum Herbst. when combined with high temper- ature 42°C Soderstrom et al., 1992. Mortality
of third instar larvae of A. ludens was significantly higher at 44°C in 1 kPa O
2
than in air Shellie et al., 1997. A 100 mortality of third instar larvae
of A. ludens was accomplished in 3.5 h in 1 kPa O
2
at 44°C Shellie et al., 1997. The most heat resistant life stage of A. ludens Loew. was re-
ported to be the third instar stage Shellie et al., 1997. In a preliminary study Yahia et al., 1997
we reported that 0 kPa O
2
+ 50 kPa CO
2
at 44°C for 160 min caused 100 mortality of eggs and
third instar larvae of A. ludens and A. obliqua. This atmosphere for 120 min caused 100 mor-
tality of larvae, but only 62 – 72 mortality in eggs of A. ludens.
The objective of this study was to evaluate the in vitro mortality of eggs and third instar larvae
of A. ludens and A. obliqua at high temperatures 44, 48, 51, 52, 54 and 55°C, in air or in CA O
2
concentration as low as 0 kPa and CO
2
concentra- tion as high as 50 kPa, for 80, 160, 220 or 240
min.
2. Materials and methods
2
.
1
. Insects handling Naked third instar larvae and eggs 50 devel-
opment of A. ludens and A. obliqua used in our experiments were obtained from the Direccio´n
General de Sanidad Vegetal-SAGAR, of the Moscafrut program in Chiapas, Mexico. Eggs
were collected for 8 h, transported the next day in water at room temperature for 8 h, and at their
arrival to the laboratory they were incubated at 25 – 27°C and 60 – 70 RH overnight. Larvae were
reared on artificial diet Planta Moscafrut, 1996 at 27°C, transported to the laboratory in the same
diet when they had 7 days of development from oviposition, and were used on the 8th day which
corresponded to the third instar stage Leyva, 1988.
At least 240 eggsreplicate were spread, using paint brush, on top of a wet filter paper over a
sponge, and placed in 250 ml plastic cups dimen- sions: 10 cm diameter, 5 cm deep. The containers
were covered with a fine mesh. After treatments, containers were cooled with water at room tem-
perature for 30 min and eggs were transferred to a petri dish containing 50 ml of distilled water,
and incubated at 25 – 27°C and 60 RH. Mortal- ity was calculated after every 24 h for 96 h or
until the chorion of the eggs that did not complete their development was interrupted. Eggs were
considered alive when they completed their em- bryonic development, characterized by the pres-
ence of the mandibles that characterize the emerging larvae.
Third instar larvae at least 240 larvaerepli- cate were deposited in 50 g of diet in a similar
container used for eggs, to which 50 ml of distilled water was added. The water was only enough to
wet the filter paper and the sponge and to main- tain high humidity during heat treatment. The
cups were covered with a mesh to prevent the escape of the larvae. After treatments, the larvae
in containers were cooled immediately with water at ambient temperature for 30 min. Larvae were
then incubated at 25 – 27°C and 60 RH, and mortality was determined after 24 h from the
treatment. Mortality was corrected using Abbott’s formula Abbott, 1925. Each larva was scored as
dead, alive, or pupated until it either pupated or died. Survived larvae where those who conserved
their mobility. Dead larvae were deposited in distilled water and observed again after 24 h.
Mortality was recorded as failure of the larvae or pupae to emerge as adults.
2
.
2
. Heat and CA treatments Eggs and third instar larvae of both species
were exposed to 21 treatments of heat either in air or in CA. Air treatments were at 44°C for 160
min, 48°C for 220 min, 51°C for 240 min, 52°C for 240 min, 54°C for 240 min, and 55°C for 240
min. Three CA regimes were applied: 0 kPa O
2
at 44°C for 160 min, 13 kPa O
2
+ 20 kPa CO
2
at 44°C for 160 min, and 0 kPa O
2
+ 50 kPa CO
2
at several temperatures and time duration. These
were 44°C for 160 min, 48°C for 220 min, 51°C for 240 min, 52°C for 240 min, 54°C for 240 min,
55°C for 240 min, 44°C for 80 min, 44°C for 160 min, 44°C for 240 min, 48°C for 80 min, 48°C for
160 min, 48°C for 240 min, 55°C for 80 min, 55°C for 160 min, and 55°C for 240 min. Relative
humidity was 50 in all treatments. Each treat- ment was accompanied with a non-treated control
in which the insects were maintained all the time in an incubator at 25 – 27°C and 60 RH.
Treatments with heat and CA at high tempera- ture were conducted inside a gas tight, tempera-
ture controlled, forced-air chamber Yahia et al., 1997. The chamber 155.8 cm high, 70 cm wide,
and 132.1 cm deep is constructed from stainless steel sheet metal. The O
2
analyzer is an electro- chemical type that provides readings over a range
of 0 to 25 kPa, and controlled within 0.1 kPa by injecting airO
2
or N
2
as required. Two N
2
se- lenoid valves provide two-stage control for stabi-
lizing the O
2
level after sealing the chamber. The infrared CO
2
analyzer provides readings between 0 and 80 and controls the environment within
0.1 kPa by injecting CO
2
or N
2
as required. If the level of O
2
rises above a set point, the system energizes the N
2
selenoid, allowing N
2
to flow into the chamber and to drive down the O
2
to the desired level. Each selenoid operates separately
depending on demand. When N
2
is needed, as a first level of response, the system energizes the N
2
selenoid that leads to the humidification nozzles. If this does not provide enough N
2
to meet the demand, the second N
2
selenoid leading to the air duct is energized. The two CO
2
selenoids operate on the same principles. The O
2
and CO
2
analyzers are calibrated routinely using the following cali-
bration gases: 100 N
2
Infra, 2.09 + 39.54 CO
2
+ 58.37 N
2
Aga, 4.99 O
2
+ 5.1 CO
2
+ 89.9 N
2
Aga, and 9.95 O
2
+ 9.98 CO
2
+ 80.07 N
2
Praxair. Chamber temperature is elevated above room temperature by means of an
electric strip heater that is energized using time proportional control, and temperature is main-
tained within 9 0.1°C over the range of 20 – 60°C by automatically energizing four 1000 W finned,
230 V electric heater elements as required. Hu- midity is provided through four atomizing noz-
zles, each has two ports; one for compressed gas and one for water. When the compressed gas
passes through the nozzle it draws distilled water from the humidification water reservoir into the
nozzle using the venturi effect. When more hu-
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
