Mortality of eggs and third instar larvae of
Anastrepha
ludens
and
A
.
obliqua
with insecticidal controlled
atmospheres at high temperatures
Elhadi M. Yahia *, Dora Ortega-Zaleta
DIPA,Facultad de Quı´mica,Uni6ersidad Auto´noma de Quere´taro,Quere´taro76010,Mexico
Received 15 November 1999; accepted 18 June 2000
Abstract
The in vitro mortality of eggs and third instar larvae of Anastrepha ludens andA.obliquawas determined after exposure to 21 treatments of air or controlled atmospheres (CA) at high temperatures and 50% RH. Air at 44°C for 160 min caused very low mortality, which increased significantly by CA. Higher temperatures caused a more rapid kill. One hundred percent mortality was achieved for third instar larvae of both species in air or CA at 48°C for 220 min. A 100% mortality of eggs ofA.ludenswas achieved in air at 51°C for 240 min or in CA at 52°C for 240 min, and 100% mortality of eggs ofA.obliquawas achieved in air or in CA at 55°C for 240 min.A.obliquawas slightly more tolerant than A. ludens, and eggs were more tolerant than third instar larvae in both species. CA had a synergistic effect at B50°C, but was slightly less effective than air at higher temperatures. Low O2 concentrations
were more effective than high CO2levels. The mean estimated temperatures for 50, 99 and 99.9968% mortality (LT50s,
LT99s, LT99.9968s) of eggs ofA.obliqua(the most tolerant) exposed to 0 kPa O2+50 kPa CO2for 240 min were 49.4,
54.8 and 60.9°C, respectively. We conclude that dry hot air at]44°C and 50% RH in CA (0 kPa O2+50 kPa CO2),
for 160 min or longer, is effective in increasing mortality of eggs and third instar larvae ofA.ludensandA.obliqua. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Postharvest; Heat; Insects; Fruit fly; Disinfestation; Quarantine; Hot air
www.elsevier.com/locate/postharvbio
1. Introduction
Mango in Mexico can be infested with fruit flies, the most important being Anastrepha ludens (Loew) andA.obliqua (L.). A disinfestation treat-ment may be required when fruit is transported to
national or export markets free of the fly. For example, all mango fruit exported to USA and Japan are treated with hot water (the only autho-rized treatment), that consists of 46.1°C for 65, 75 or 90 min (depending on fruit weight). This treat-ment, especially for longer periods, accelerates ripening and causes fruit injury (Campos and Yahia, 1991). Modified (MA) or controlled atmo-spheres (CA) can be lethal to some insects and yet
* Corresponding author. Tel./fax: +52-42-156867. E-mail address:[email protected] (E.M. Yahia).
beneficial for preserving the postharvest quality of several fruits (Yahia, 1998). The lethal MA/CA 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 differdiffer-ent crops. Some mango culti-vars such as ‘Keitt’ are tolerant to insecticidal (extreme) atmospheres (0.1 – 0.5 kPa O2 and/or
50 – 80 kPa CO2) 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 and/or 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 N2killed 7.4% of larvae (Benschoter
et al., 1981). Sixty hours of exposure to 100 kPa N2 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 O2
(1 kPa) at high temperatures increased the mortal-ity of several insects. The use of high levels of CO2 (60 – 90 kPa) and low levels of O2 (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 ofA.ludenswas significantly higher at 44°C in 1 kPa O2than 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 O2 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 O2+50 kPa CO2 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% mormor-tality 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 (O2
concentration as low as 0 kPa and CO2
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).
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 larvae/ repli-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 O2(at
44°C for 160 min), 13 kPa O2+20 kPa CO2 (at
44°C for 160 min), and 0 kPa O2+50 kPa CO2at
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 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 O2 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 air/O2 or N2 as required. Two N2
se-lenoid valves provide two-stage control for stabi-lizing the O2 level after sealing the chamber. The
infrared CO2 analyzer provides readings between
0 and 80% and controls the environment within 0.1 kPa by injecting CO2or N2as required. If the
level of O2 rises above a set point, the system
energizes the N2selenoid, allowing N2to flow into
the chamber and to drive down the O2 to the
desired level. Each selenoid operates separately depending on demand. When N2 is needed, as a
first level of response, the system energizes the N2
selenoid that leads to the humidification nozzles. If this does not provide enough N2 to meet the
demand, the second N2selenoid leading to the air
duct is energized. The two CO2 selenoids operate
on the same principles. The O2and CO2analyzers
are calibrated routinely using the following cali-bration gases: 100% N2 (Infra), 2.09%+39.54%
CO2+58.37% N2(Aga), 4.99% O2+5.1% CO2+
89.9% N2 (Aga), and 9.95% O2+9.98% CO2+
80.07% N2 (Praxair). Chamber temperature is
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 m/s and air velocity inside the chamber averaged 2.5 – 3.5 m/s (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. LT50, LT99 and LT99.9968 (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 O2+50 kPa CO2at 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 O2 and CO2 recorded
during a treatment of 0 kPa O2+50 kPa CO2 at
48°C for 220 min is shown in Fig. 1. Temperature and CO2 increased rapidly, while O2
concentra-tion decreased rapidly in the chamber. Depending on the temperature/time 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 O2
concentration in 15 – 20 min, and the target CO2 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 O2
(0 kPa) than with the use of high CO2
concentra-tion (20 kPa). The low O2 (0 kPa) and high CO2
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
Table 1
Corrected mortality (%) of eggs and third instar larvae ofA.ludensandA.obliqueat different atmospheres (kPa O2–kPa CO2), temperatures (°C) and exposure times (min)a
Temperature
Atmosphere Time (min) A.ludens A.obliqua
(°C)
aValues in parenthesis indicate standard error of the mean
eggs ofA. obliqua, which had an average mortal-ity of 63.4%. The mortalmortal-ity at 54°C was somewhat similar to that achieved at 52°C, except that there was a slight increase in the mortality of eggs ofA. obliquain 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 CO2 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 ofA.obliquaafter 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 548°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 LT50s,
LT99s, and LT99.9968s of eggs of A. obliqua (the
most tolerant stage of both insects) exposed to 0 kPa O2+50 kPa CO2 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).
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 ofA.obliquathan in air, although not statistically different. The toxicity of CA for insects, especially the higher concentration of CO2 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 CO2and/or lower concentration of O2, as it is the
case during the development of some stages of the insect inside the fruit. Because CO2 hydrate is
carbonic acid, it must produce acidification and act indirectly on the membranes by modifying their permeability (Nicolas and Sillans, 1989). Mixtures of CO2 and air may be more rapidly
lethal to some species than pure CO2 (Baily and
Banks, 1975, 1980). Lethal effects of CO2 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 CO2 seems necessary
since the off-spring of the survivors may show increased resistance, which would necessitate longer exposure periods to the same concentration of CO2 to obtain similar mortality (Bond and
Buckland, 1979; Navarro et al., 1985).
Soderstrom et al. (1990) have found that low O2
(0.5 kPa) is less effective than high concentration of CO2 in the mortality of codling moth at 25°C.
However, Shellie et al. (1997) using very few insects found that reduced O2 concentration (1
kPa) was more lethal toA.ludenslarvae than was an enriched CO2atmosphere (20 kPa), which is in
agreement with what we are reporting here onA. ludens and A. obliqua. The larval mortality of both species was higher in low O2 than in high
CO2 concentration (Table 1).
Our results indicated that eggs ofA.ludensand 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 O2 deficient and CO2
en-riched atmospheres. Fifth instar larvae was re-ported to be the most tolerant life stage of Epiphyas post6ittana to 0.4 kPa O2 with 5 kPa CO2 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 pomonellaeggs were equally suscep-tible to CA with O2 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 O2+50 kPa CO2at 51, 54 and 55°C for
an, number of treatments;m9S.E., slope9standard error;
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 O2 or CO2 atmosphere
composition.
4. Conclusions
We conclude that the use of CA (0 kPa O2with
or without 50 kPa CO2) at high temperatures (44,
48, 51, 52 and 54°C) for 80, 160 or 240 min caused high mortality of eggs and third instar larvae ofA.ludensandA.obliqua, compared with air at the same temperature and duration. Higher temperatures caused a more rapid kill, and CA had a synergistic effect at temperatures B50°C. There seem to be no benefits in using CA at temperatures ]50°C. A 100% mortality was achieved at ]48°C for larvae and ]51°C for eggs. Eggs were more tolerant than third instar larvae in both species, and eggs ofA. obliqua are slightly more tolerant than those of A. ludens. Low O2 concentrations were more effective than
high CO2 concentrations. Temperatures higher
than 44°C for 160 min or longer can cause injury to some mango cultivars such as ‘Manila’ (Yahia et al., 1997). The efficiency of these treatments are being confirmed in our laboratory with in vivo studies using a greater number of insects.
Acknowledgements
We thank Dr J.L. Leyva-Vazquez from Colegio de Posgraduados, Montecillos and Alejandro Martinez and Pamela Moreno from DGSV-SAGAR, Tapachula, Chiapas for supplying the insects.
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