Resistance of Mexican Fruit Fly to Quarantine Treatmentsof High-Pressure Processing Combined with Cold

Juan F. Castanon-Rodrıguez,1 Manuel A. Vargas-Ortiz,2 Pablo Montoya,3 Hugo S. Garcıa,1

Gonzalo Velazquez,1,4 Jose A. Ramırez,1 and Manuel Vazquez1,5

Abstract

Mexican fruit fly Anastrepha ludens Loew (Diptera: Tephritidae) is one of the most important insects infestingfruits. Although high pressure has been proposed as an alternative quarantine process for this pest, conditionsapplied to destroy eggs and larvae can also damage the fruits. The objective of this study was to assess thebiological viability of A. ludens eggs treated by high-pressure processing at 08C, establishing whether non-destroyed eggs and larvae preserved their ability to develop and reproduce. One-, 2-, 3-, and 4-day-old eggswere pressurized at 50, 70, or 90 MPa for 0, 3, 6, or 9 min. The hatching ability of pressurized eggs and theircapacity to develop larvae, pupae, and reproductive adults were evaluated. The ability of pressurized larvae topupate was also registered. The results showed that most of the eggs that resisted the treatments were able toproduce adults with capability to reproduce a new generation of A. ludens flies. Larvae were less resistant thaneggs. All larvae were killed at 90 MPa for 9 min. The pressure induced the expression of heat shock proteins(HSP) in second- and third-instar larvae. The HSP showed a baroprotective effect in A. ludens larvae. Theseresults are relevant to the industry because they show that eggs of insects infesting fruits treated by high-pressure processing were able to pupate after treatments. This indicates that efforts must be addressed to destroyall eggs because most of the surviving organisms could reach an adult stage and reproduce.

Introduction

Fruit flies are considered as one of the critical quaran-tine problems for the international marketing of fruits. To

avoid the introduction of these pests, many countries requirea postharvest sanitation treatment for imported fruits (Gazitet al., 2004). Approved treatments include fumigation withtoxic compounds, irradiation, and physical treatments in-cluding heating and cooling (Armstrong and Mangan, 2007).

Cold storage has been used as a quarantine method sincethe beginning of the 20th century (Mangan and Hallman,1998). USDA-APHIS has approved holding fruit at 1.18C, orless, for 12 days as a cold treatment to be used against thisspecies for 17 commodities from 49 countries (Phillips et al.,1997; Gazit et al., 2004).

Although cold treatment was approved as a quarantinemethod, it has been reported that it can reduce the commercialvalue and quality of many tropical fruits because of chillinginjury (Phillips et al., 1997; Gazit et al., 2004; Lanza et al., 2005),which imposes a practical limitation to select cold treatment

as a quarantine method (Lederman et al., 1997; Mangan andHallman, 1998).

The use of high-pressure processing (HPP) has been re-ported as an alternative quarantine process for Mediterraneanfruit fly, Mexican fruit fly, western cherry fruit fly, and cod-ling moth (Butz and Tauscher, 1995; Neven et al., 2007;Candelario-Rodrıguez et al., 2009; Velazquez et al., 2010a,2010b); however, early studies have found that the HPPconditions required to destroy the eggs and larvae of thesepests caused unacceptable damage to the fruits.

The use of combined treatments to meet quarantine re-quirements has been reviewed for a number of combinedapproaches (Mangan and Sharp, 1994). Using cold tempera-tures combined with HPP has been reported as an effectivealternative to destroy eggs and larvae of Mexican fruit fly(Hernandez-Ortiz, 1992; Velazquez et al., 2010b). However,pressurization levels above 100 MPa can induce physical andphysiological changes in the targeted fruit (Neven et al., 2007;Candelario-Rodrıguez et al., 2009). On the other hand, it isimportant to consider that profitability of HPP requires short

1Department of Food Science and Technology, UAM Reynosa Aztlan, University Autonoma de Tamaulipas, Reynosa, Tamaulipas, Mexico.2UNIDA, Technological Institute of Veracruz, Veracruz, Mexico.3Unit of Methods Development, Program Moscafrut SAGARPA-IICA, Tapachula, Chiapas, Mexico.4CICATA Queretaro, Queretaro, Mexico.5Department of Analytical Chemistry, Faculty of Veterinary Science, University of Santiago de Compostela, Lugo, Spain.

FOODBORNE PATHOGENS AND DISEASEVolume 8, Number 7, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089=fpd.2010.0811

815

processing times (Velazquez et al., 2005a, 2005b). Therefore,the operating conditions of HPP to destroy eggs and larvaeshould be less than 100 MPa and short times (less than10 min).

On the other hand, heat shock proteins (HSP) act in theorganisms as molecular chaperones (Craig et al., 1993).These proteins protect the organisms from injury at hightemperatures by avoiding protein denaturation. HSPappear to be capable of recognizing simple structuralmotifs shared by many unfolded proteins (Denlinger andYocum, 1998). HSP have been reported to be expressedduring high-pressure treatments of yeast and bacteria,protecting microorganisms against pressure damage.However, it has not been reported in fruit fly as a responseagainst pressure.

The objectives of this work were to determine the com-bined effect of HPP and cold temperature (08C) on the capa-bility of Mexican fruit fly eggs and larvae to survive andpreserve their biological capacity to develop and reproduceand, second, to elucidate whether HPP can induce the pro-duction of HSP in Mexican fruit fly as a possible protectingmechanism.

Materials and Methods

Sampling site

The experimental work was conducted at the facility ofMoscafrut in Metapa de Domınguez, Chiapas, Mexico, whichproduces Anastrepha ludens for sterile insect release programs.The eggs and larvae used in this study were viable (non-sterilized), supplied by this facility, and reared as reportedelsewhere (Dominguez et al., 2010).

High-pressure system

Pressure treatments were carried out as reported elsewhere(Candelario et al., 2010). Briefly, a mixture of 5:1 water withcrushed ice:anticorrosive lubricant (Hydrolubric 120-B; EFHoughton, Valley Forge, PA) was used as pressurizing fluidto maintain a temperature near 08C during treatments. Thetemperature of the fluid was never higher than 08C as the icewas still present at the end of the treatments.

Eggs and larvae were pressurized at 50–90 MPa for 0–9 minat 08C. The time required to reach process pressure was lessthan 44 sec and the release time was always less than 30 sec. Atreatment for 0 min is a treatment wherein the pressure isreached and released immediately.

Larvae for studies on polyacrylamide gel electrophoresis(see below) were treated at 50–150 MPa for 0–20 min.

High pressure treatments on eggs

One-, 2-, 3-, or 4-day-old eggs were processed inde-pendently in two runs. A 4.5 mL aliquot of an egg=watersolution containing 24,200 eggs=mL (108,900 total eggs)was taken from a 10-L plastic container and placed intothree Eppendorf � tubes with 2 mL capacity. The tubeswere filled completely with water, closed immediately, andpressurized.

High-pressure treatments on larvae

The same high-pressure treatments were applied to 30,000larvae of each instar. Briefly, larvae were manually sepa-rated, weighed, and counted using a small quantity of talcpowder to facilitate handling. The number of larvae in 5 g (bytriplicate) was used to estimate the amount required to ob-tain 30,000 larvae. Larvae were introduced into plasticpouches of 200 mL and sealed immediately before the high-pressure treatment. After treatment, the surviving larvaewere transferred in groups of 100 larvae to Petri dishescontaining vermiculite and held at 208C and 80% relativehumidity in the dark for 48 h. After the holding period, thenumber of pupae formed was recorded. The survivorship toemergence or reproductive capacity of the flies was notregistered.

Determination of eggs hatching

After pressurizing treatments, the eggs from one Eppen-dorf tube were extracted using a brush and arranged in threerows of 100 eggs in Petri dishes using a Zeiss Stemi SV 6stereoscopic microscope and incubated at 268C� 18C for 7days to monitor egg hatching. Three replicates of this bioassaywere performed.

Table 1. Effect of Time and Level of Pressurizing at 08C on the Inhibition of Anastrepha ludens Egg Hatch

Pressure (MPa) Time (min) Day 1 (%) Day 2 (%) Day 3 (%) Day 4 (%)

0 (Control) 0 92.0� 2.8a 93.9� 1.5a 91.8� 1.8a 95.5� 1.0a

50 0 86.6� 4.6a 88.8� 3.3a 89.0� 2.3a 90.4� 2.2a

50 3 73.0� 6.6b 77.2� 3.6b 73.0� 6.6b 25.6� 4.8b

50 6 54.6� 3.9c 70.0� 5.6b 58.5� 2.4b 27.6� 5.9b

50 9 37.7� 3.0d 51.0� 5.4c 6.8� 2.6c 16.7� 2.5c

70 0 21.0� 2.9a 77.6� 4.0a 87.0� 2.6a 72.8� 3.8a

70 3 0.0� 0.0b 1.2� 1.2b 35.7� 2.7c 18.6� 5.0c

70 6 0.0� 0.0b 0.0� 0.0b 45.8� 2.7b 26.3� 3.7b

70 9 0.0� 0.0b 0.0� 0.0b 19.8� 3.8d 14.4� 2.7c

90 0 3.3� 1.6a 28.5� 3.1a 20.2� 3.3a 22.7� 2.7a

90 3 0.0� 0.0b 3.8� 2.2b 15.7� 2.9b 19.2� 1.5a,b

90 6 0.0� 0.0b 0.0� 0.0c 10.4� 1.1c 15.0� 3.0b

90 9 0.0� 0.0b 0.0� .0.0c 8.7� 5.1c 0.0� 0.0c

Mean values and standard deviations of two studies with two replicates are shown.a–dDifferent letters in the same row indicate significant differences ( p< 0.05) between time of processing for each level of pressurizing.

816 CASTANON-RODRIGUEZ ET AL.

Determination of surviving eggs’ capabilityto form larvae, to pupate, and to produce adultsand the reproductive capacity of emerged adults

These determinations were performed as reported else-where (Candelario et al., 2010).

Microphotography of eggs

Nonhatching eggs were photographed using a digitalcamera (Nikon DXM1200F) attached to a dissecting micro-scope (Nikon SM21500).

Electrophoresis

One-dimensional sodium dodecyl sulfate–polyacrylamidegel electrophoresis was performed according to Laemmli(1970). In all cases, a 4% stacking gel and a 12% separating gelwere used. Electrophoresis was performed at 20 mA per gel.An aliquot of molecular weight (MW) standard mixture(MW.SDS.70L; Sigma Chemical) was included in all gels.

Statistical analysis

Statistical analysis was performed using a Statgraphics 5.0(Software Publishing Corporation, Bitstream, Inc.). In egg’s

FIG. 1. Anastrepha ludens eggs pressurized at 0 (control), 50, 70, or 90 MPa for 9 min at 08C.

RESISTANCE OF MEXICAN FRUIT FLY TO QUARANTINE TREATMENTS 817

bioassay, LSD’s multiple range tests were used to determinesignificant differences ( p< 0.05) between treatments.

Results

Effect of high pressure on eggs

The effect of the pressurizing level and treatment time isshown in Table 1. The hatching ability of eggs was affected byboth variables, time and pressure level.

The hatching ability of 1- and 2-day-old eggs was moresensitive to this treatment than the 3- and 4-day-old eggs. All1-day-old eggs completely lost their ability of hatchingby using 70 or 90 MPa for 3 and 6 min. However, some of the3-day-old eggs were able to hatch even after 90 MPa for 9 min.

Figure 1 shows the effect of HPP on the inner structure of A.ludens eggs that were not able to hatch.

Development of pressurized eggs to form larvae,pupae, and adults

The results for the ability of pressurized eggs to form larvaeare shown in Table 2. The rate of larvae formed from pres-surized eggs that survived until third-instar stage was af-

fected by time and pressure level. Nonpressurized controleggs showed more than 91% of larvae survival.

The number of eggs that were able to hatch at 50 and70 MPa for 0 min for 2-day-old eggs (Table 1) were slightlylower than the number of surviving larvae reported in Table 2.This difference was most likely caused because data entries inTables 1 and 2 represent an approximation obtained bypopulation estimations. Table 1 reports an estimate obtainedfrom the rate of hatching of 300 eggs (by triplicate), whereasdata of Table 2 were obtained by counting the number oflarvae in 5 g, and such data were employed to calculate thetotal number of larvae.

The effect of HPP on the ability to pupate of survivinglarvae was determined and results are shown in Table 3. Mostof the surviving eggs were able to form third instars (Table 2)with enough vitality to develop until the pupae stage wasreached (Table 3). The ability to form pupae decreased at aminimum when eggs were pressurized at 90 MPa. Even at90 MPa for 9 min, some pressurized eggs were able to reachthe pupae stage.

Most of the eggs that were able to survive the treatmentsand preserved their ability to form pupae (Table 3) preservedalso their ability to form adults (Table 4).

Table 2. Percentage of Larvae Obtained from the Pressurized Eggs of Anastrepha ludens

at Each Level and Time of Pressurizing (Initial Eggs¼ 108,900)

Pressure (MPa) Time (min) Day 1 (%) Day 2 (%) Day 3 (%) Day 4 (%)

0 (Control) 0 91.4� 0.1 97.1� 4.4 97.8� 2.4 95.5� 6.550 0 14.0� 1.2 91.4� 5.8 88.9� 13.2 90.9� 5.350 3 11.0� 3.5 28.6� 1.2 44.4� 1.4 0.2� 1.250 6 2.9� 0.2 7.1� 0.1 22.2� 0.5 5.5� 0.250 9 0.0� 0.0 4.3� 2.0 17.8� 2.7 0.4� 3.470 0 7.1� 3.3 84.3� 4.3 66.7� 0.5 36.4� 2.370 3 0.0� 0.0 1.4� 2.9 15.6� 3.2 0.5� 0.570 6 0.0� 0.0 1.4� 0.4 11.2� 0.4 0.1� 0.670 9 0.0� 0.0 2.9� 0.1 3.3� 0.1 0.5� 0.390 0 2.0� 2.4 20.0� 3.0 15.6� 0.5 1.0� 0.490 3 0.0� 0.0 2.9� 0.6 8.9� 1.6 0.3� 0.0490 6 0.0� 0.0 0.0� 0.0 8.6� 0.0 0.6� 0.290 9 0.0� 0.0 0.4� 0.1 4.4� 2.0 0.0� 0.9

Mean values and standard deviations of two studies with two replicates are shown.

Table 3. Effect of Time and Level of High Pressure at 08C on Pupation of Surviving

Anastrepha ludens Larvae from Pressurized Eggs

Pressure (MPa) Time (min) Day 1 (%) Day 2 (%) Day 3 (%) Day 4 (%)

0 (Control) 0 85.6� 1.2 95.7� 2.4 90.3� 1.5 88.8� 3.150 0 10.4� 3.0 85.2� 3.6 82.6� 8.8 90.9� 5.350 3 8.7� 1.4 27.4� 0.0 44.4� 1.3 0.2� 0.550 6 2.0� 0.1 7.1� 0.1 20.8� 0.8 4.8� 1.050 9 0.0� 0.0 4.0� 0.1 13.4� 1.0 0.4� 2.370 0 5.7� 0.2 81.6� 0.7 66.7� 0.5 32.9� 2.070 3 0.0� 0.0 1.4� 2.9 12.6� 1.3 0.4� 0.270 6 0.0� 0.0 1.2� 0.1 11.0� 1.9 0.1� 0.670 9 0.0� 0.0 1.7� 0.1 3.3� 0.1 0.4� 1.290 0 1.9� 0.2 20.0� 3.0 13.9� 2.5 1.0� 0.290 3 0.0� 0.0 1.6� 0.3 8.0� 0.7 0.3� 0.090 6 0.0� 0.0 0.0� 0.0 8.5� 0.6 0.5� 1.490 9 0.0� 0.0 0.3� 0.0 4.0� 0.1 0.0� 0.9

Mean values and standard deviations of two studies with two replicates are shown.

818 CASTANON-RODRIGUEZ ET AL.

The adults obtained from pressurized eggs were matchedwith individuals of their opposite sex to determine whetherthey retained their reproductive ability and whether the neweggs were able to hatch (Table 5). Flies obtained from pres-surized eggs were able to reproduce and their eggs were ableto hatch.

Effect of high pressure on larvae

Larvae showed very low resistance to HPP even at 50 MPa(Table 6). The surviving rate decreased drastically with in-creasing time and pressurizing level. Third instars showed thehighest resistance to HPP. However, larvae from all instarsdid not survive when pressurized at 90 MPa for 9 min. A highrate of surviving larvae preserved their ability to pupate(Table 7).

Expression of HSP

The expression of HSP by pressurized second and thirdinstars is depicted in Figures 2 and 3, respectively. Larvaewere pressurized at 50–150 MPa for 0–20 min. Second instarsshowed the presence of proteins with MW greater than

66 kDa, corresponding to the HSP70 family. HSP of low MWwere also expressed in a very low concentration (Fig. 2). Thirdinstars showed a high concentration of the HSP70 family. HSPof low MW were also observed but in lower concentrations.

Discussion

Effect on eggs hatching

It was reported that HPP at 50 MPa and 258C showed aminimum effect on the eggs hatching of A. ludens even after20 min. The effect of pressurizing at 50 MPa was improved bydecreasing the initial temperature to 08C (Velazquez et al.,2010b). However, in such study, the final process temperaturefluctuated between 08C and 18C and the hatching level rangedbetween 22% and 82%, depending on the age of the eggs. Inour study, a greater inhibition of eggs hatching was observedat 50 MPa (Table 1), because the temperature of the pressuri-zation chamber was held constant at 08C during the entireprocess. These results suggest the importance of temperaturecontrol on the lethal effect of HPP.

Some eggs showed higher resistance to HPP and preservedtheir ability to hatch. This resistance could be associated with

Table 4. Percentage of Adults Obtained from the Pressurized Eggs of Anastrepha ludens

at Each Level and Time of Pressurizing (Initial Eggs¼ 108,900)

Pressure (MPa) Time (min) Day 1 (%) Day 2 (%) Day 3 (%) Day 4 (%)

0 (Control) 0 82.2� 0.9 90.2� 1.0 86.2� 2.0 84.3� 1.650 0 9.6� 1.2 82.3� 2.2 80.8� 7.6 90.9� 5.350 3 7.7� 0.2 23.0� 0.7 44.0� 0.9 0.2� 0.550 6 1.0� 0.1 7.1� 0.1 17.5� 1.0 4.6� 1.050 9 0.0� 0.0 3.8� 0.2 9.9� 0.0 0.4� 2.370 0 5.7� 0.2 78.4� 1.2 64.6� 1.7 30.2� 1.070 3 0.0� 0.0 1.0� 2.0 10.2� 1.0 0.4� 0.170 6 0.0� 0.0 1.2� 1.0 10.9� 0.7 0.1� 0.670 9 0.0� 0.0 1.6� 0.1 3.0� 0.1 0.3� 0.990 0 1.0� 1.0 18.4� 2.6 11.9� 3.0 0.9� 1.290 3 0.0� 0.0 1.5� 0.0 7.8� 1.0 0.3� 0.090 6 0.0� 0.0 0.0� 0.0 7.9� 1.0 0.5� 0.990 9 0.0� 0.0 0.1� 0.0 3.9� 0.1 0.0� 0.9

Mean values and standard deviations of two studies with two replicates are shown.

Table 5. Percentage of Hatching of Eggs of Anastrepha ludens Oviposited for Adults Obtained

from the Pressurized Eggs at Each Level and Time of Pressurizing

Pressure (MPa) Time (min) Day 1 (%) Day 2 (%) Day 3 (%) Day 4 (%)

0 (Control) 0 93.1� 3.2 89.0� 9.8 93.2� 6.8 91.6� 3.650 0 79.3� 13.2 90.2� 14.3 85.3� 2.9 88.0� 10.550 3 53.9� 7.9 76.5� 4.5 80.3� 4.5 53.0� 3.250 6 43.9� 1.6 65.7� 3.0 74.3� 1,2 59.3� 3.850 9 — 46.7� 3.4 70.4� 8.0 50.2� 1.470 0 78.3� 10.0 83.4� 11.0 89.0� 2.3 77.8� 15.070 3 — 65.3� 7.8 77.9� 14.5 42.0� 1.470 6 — 61.3� 3.2 80.5� 4.7 37.2� 2.970 9 — 67.1� 4.9 64.3� 1.9 48.3� 2.090 0 39.5� 1.0 72.1� 1.8 81.0� 4.6 57.3� 1.690 3 — 59.7� 2.2 63.2� 1.9 38.4� 5.090 6 — — 60.9� 5.6 40.6� 9.290 9 — 39.0� 3.2 59.4� 3.4 30.6� 7.4

Mean values of one study with three replicates are shown.

RESISTANCE OF MEXICAN FRUIT FLY TO QUARANTINE TREATMENTS 819

the physiology of the eggs, because HPP is instantaneous anduniform (Tellez-Luis et al., 2001; Torres and Velazquez, 2005;Velazquez et al., 2005a, 2005b).

One- and 2-day-old eggs pressurized at 50–90 MPa for9 min showed a change in the inner structure, compared withthe control eggs (Fig. 1). The changes were more evident athigher pressures. Eggs are composed mainly of water andproteins. The effect of HPP on egg hatching has been associ-ated with a change in the structure and permeability of ex-ternal and internal membranes, denaturation=aggregation ofproteins, or a combination of both factors (Borderıas et al.,1997; Fernandez-Martın et al., 1998; Uresti et al., 2004; Ve-lazquez et al., 2010b).

In this study, it was observed that HPP induced, in 1-day-old eggs, the flocculation and separation of proteinaceous

structure, compared with control eggs (Fig. 1). This behaviorwas more evident when the pressure was increased until a fulldisruption of the cellular membrane was achieved at 90 MPafor 9 min. A similar behavior was observed for 2-day-old eggs,although these eggs did not show the rupture of the cellularmembrane.

The loss of hatching ability in eggs induced by HPP at 08Ccould be due to protein denaturation=aggregation, becausethis phenomenon affects the viability of the organism (Farr,1990; Pothakamury et al., 1995; Tellez-Luis et al., 2001).

Although most eggs lost their hatching ability, some eggswere able to survive and hatch. This resistance has been re-ported in some specimens for other quarantine treatmentssuch as cold or heat shock injury as well as long-term heatingand chilling injury (Denlinger and Lee, 1998; Denlinger andYocum, 1998; Ashie et al., 1999; Uresti et al., 2005).

Surviving larvae obtained from pressurized eggs

The rate of surviving larvae reaching the third instar (Table2) was lower than the percentage of hatched eggs (Table 1).This difference may be associated with physicochemicalchanges caused by the pressurizing treatment on proteinsinvolved in DNA replication and transcription (Farr, 1990;Pothakamury et al., 1995; Tellez-Luis et al., 2001). Such chan-

Table 6. Effect of Time and Level of Pressure at 08Con Survival of Anastrepha ludens Larvae

Surviving larvae (%)

Pressure(MPa)

Time(min)

Firstinstars

Secondinstars

Thirdinstars

0 (Control) 0 95.7 96.3 98.750 0 3.5 24.9 42.0

3 2.4 6.2 32.36 0.0 4.8 26.59 0.0 8.9 3.7

70 0 2.3 4.6 21.43 1.3 2.2 5.36 0.0 1.1 11.69 0.0 0.2 0.0

90 0 1.7 0.0 26.43 0.0 0.4 15.86 0.0 0.0 2.69 0.0 0.0 0.0

Mean values of one analysis for approximately 30,000 larvae areshown.

Table 7. Effect of Time and Level of High Pressure

at 08C on Pupation of Surviving Anastrepha

ludens Larvae

Pupating (%)

Pressure(MPa)

Time(min)

Firstinstars

Secondinstars

Thirdinstars

0 (Control) 0 85.0 87.0 90.350 0 2.0 9.9 32.0

3 1.2 3.7 24.06 0.0 2.5 19.69 0.0 6.2 2.3

70 0 0.8 4.2 15.23 1.2 1.0 4.86 0.0 0.6 7.89 0.0 0.2 0.0

90 0 0.9 0.0 22.13 0.0 0.4 10.66 0.0 0.0 1.79 0.0 0.0 0.0

Mean values of one analysis for approximately 30,000 larvae areshown.

FIG. 2. Electrophoretic pattern of proteins extracted fromsecond instars pressurized at different conditions. Arrowsindicate the presence of proteins expressed as a result ofhigh-pressure processing. Lane 1 shows the molecularweight marker proteins and lane 2 is a nonpressurizedsample (control).

820 CASTANON-RODRIGUEZ ET AL.

ges could induce the formation of larvae with physiologicaldeviations, which inhibit their growth and ability to reachthird instar.

Formation of pupae from surviving larvaeand adults from pupae

Although some larvae were not able to pupate, most ofthem did (Table 3), indicating that most of the surviving eggspreserved their physiological properties and biological func-tionality associated with growth. Most of the pupae emergedas adult flies, confirming that most of the surviving eggspreserved their viability.

Viability of eggs produced by adultsobtained from pressurized eggs

Most of the adults that emerged from eggs resisting HPPpreserved their ability to reproduce and lay viable eggs(Table 5). Recently, it was reported that eggs resisting HPPtreatments at initial 508C maintained their ability to repro-duce and lay viable eggs (Candelario et al., 2010). These re-sults confirm that efforts to optimize HPP as a quarantinemethod for A. ludens must be focused to destroy both eggs

and larvae, because the surviving organism could be ableto reproduce.

Effect of pressurization on larvae

HPP killed most of the larvae by inducing changes in theirexternal and inner structure as well as by modifying theirproteins. The physical damage of larvae by HPP included thepresence of dark regions, air bubbles, separation of externalmembrane, and liquid accumulation (Candelario et al., 2010).

HPP induced the expression of hsp in second and third in-stars (Figs. 2 and 3). Second instars showed a lower expressionof HSP than third instars associated with a lower resistance toHPP (Table 6). The higher resistance of the third instars to HPPseems to be associated with the expression of proteins from theHSP70 family. To our knowledge, there are no reports of thepresence of HSP in fruit flies derived from a pressurizingtreatment. All organisms respond to heat and a variety of otherstresses such as cells anoxia, ethanol, and certain heavy metalions, by inducing the synthesis of HSP. The stress proteinsencoded by the HSP70 and HSP90 gene families in response toelevated temperatures are among the most highly conservedproteins. The HSP themselves or their close relatives are pres-ent in all organisms at normal temperatures and play vital rolesin normal cell function (Lindquist and Craig, 1988).

HSP70 appears to be the most prominent contributor tothermotolerance in insects. These are the proteins that respondsmost dramatically to heat shock, and it can be induced morethan 1000-fold in response to heat shock (Velazquez et al., 1983;Denlinger and Yocum, 1998). In our study, HSP were ex-pressed, as a response to high pressure, in a high concentrationin third instars, thereby protecting them against pressurizationand increasing their surviving rate during HPP.

Effect of pressurization on expression of HSP

The small HSP that were less expressed than the HSP70family, had a modest 10-fold increase in expression but theyare important in the high temperature response (Denlingerand Yocum, 1998). During HPP the small HSP were expressedin low concentrations in both second and third instars (Figs. 2and 3), but seemed to be associated with the resistance ofsome individuals to HPP. Small HSP with an MW of 15–30 kDa are ubiquitous and conserved. These proteins act asmolecular chaperones in folding reactions and play a role inthe formation or maintenance of the native conformation ofcytosolic proteins. HSP promote the functional refolding ofproteins after denaturation.

In Saccharomyces cerevisiae, HSP provided protectionagainst subsequent pressure damage. The parallel inductionof barotolerance and thermotolerance by heat shock sug-gested that high-pressure and high-temperature effects onyeast may be physiologically tightly linked (Iwahashi et al.,1991; Jakob et al., 1993; Aertsen et al., 2004). The data found inour study are in agreement with these results. The HSPshowed a baroprotective effect on A. ludens larvae.

Conclusions

Hatching of A. ludens eggs was inhibited by pressurizing at50–90 MPa for 0–9 min at 08C. However, some eggs were ableto survive the process and preserved the ability to hatch, formlarvae, pupate, and develop into adults with reproductive

FIG. 3. Electrophoretic pattern of proteins extracted fromthird instars pressurized at different conditions. Arrows in-dicate the presence of proteins expressed as a result of high-pressure processing. Lane 1 shows the molecular weightmarker proteins and lane 2 is a nonpressurized sample(control).

RESISTANCE OF MEXICAN FRUIT FLY TO QUARANTINE TREATMENTS 821

capacity. The larvae of A. ludens were less tolerant to pressurethan eggs. All larvae died at 90 MPa for 9 min. These resultsindicate that efforts must be addressed to destroy all eggs andlarvae because most of the surviving organism could be ableto reach adult stage and reproduce.

Acknowledgments

The authors are grateful to PROMEP for financial supportto this work, to Jose Manuel Gutierrez Ruelas, Director deMoscas de la Fruta, DGSV-SENASICA SAGARPA, for logisticsupport, and to Refugio Hernandez and Emilio Hernandez(Programa Moscafrut SAGARPA-IICA) for technical andscientific advice.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Manuel Vazquez, Ph.D.

Department of Analytical ChemistryFaculty of Veterinary Science

University of Santiago de CompostelaCarballo Calero s=n

Lugo, Spain

E-mail: manuel.vazquez@usc.es

RESISTANCE OF MEXICAN FRUIT FLY TO QUARANTINE TREATMENTS 823