A r t i c l e

HIGH HYDROSTATIC PRESSURE

INDUCES SYNTHESIS OF
HEAT-SHOCK PROTEINS AND
TREHALOSE-6-PHOSPHATE
SYNTHASE IN Anastrepha ludens
LARVAE
Manuel A. Vargas-Ortiz
Unidad de Investigación y Desarrollo en Alimentos, Instituto Tecnológico
de Veracruz, M.A. de Quevedo, Col. Formando Hogar, Veracruz,
Ver. México

Rodolfo Quintana-Castro
Facultad de Bioanálisis-Veracruz, Universidad Veracruzana, Carmen
Serdan, Veracruz, Ver. México

Rosa M. Oliart-Ros and Javier De la Cruz-Medina

Unidad de Investigación y Desarrollo en Alimentos, Instituto Tecnológico
de Veracruz, M.A. de Quevedo, Col. Formando Hogar, Veracruz,
Ver. México

José A. Ramı́rez de León

Centro de Excelencia, Dirección General de Innovación Tecnológica,
Universidad Autónoma de Tamaulipas, Victoria, México

Hugo S. Garcia
Unidad de Investigación y Desarrollo en Alimentos, Instituto Tecnológico
de Veracruz, M.A. de Quevedo, Col. Formando Hogar, Veracruz,
Ver. México

The Mexican fruit fly (Anastrepha ludens) is responsible for losses of up to

25% of crops such as mango and citrus fruits in Central America and

Grant sponsor: PROMEP (Mexico).

Correspondence to: H. S. Garcia, Unidad de Investigación y Desarrollo en Alimentos, Instituto Tecnológico
de Veracruz. M.A. de Quevedo No. 2779, Col. Formando Hogar, Veracruz, Ver. 91897 México. E-mail:
hsgarcia@itver.edu.mx

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 82, No. 4, 196–212 (2013)
Published online in Wiley Online Library (wileyonlinelibrary.com).

C 2013 Wiley Periodicals, Inc. DOI: 10.1002/arch.21085
 Stress Proteins in Fruit Fly Larvae r 197

México. The larval life cycle of A. ludens comprises three stages with a
duration ranging from 3 to 8 days. Because of the damage caused by
A. ludens, several methods of control have been studied and implemented.
High hydrostatic pressures (HHP) are currently applied to foods and it is
now proposed to be employed to inactivate eggs and larvae of A. ludens.
Originally HHP was designed to inactivate microorganisms, since it exerts
marked effects on cell morphology, and can affect enzymatic reactions and
genetic mechanisms of microbial cells, with no major changes altering the
sensory or nutritional quality of the foodstuff. In this study, A. ludens in
two larval stages (5- and 8-day-old) were subjected to HHP treatments. The
biochemical response of the larvae of A. ludens was dependent on their
stage of development. The third larval stage (L3) developed a better
protection mechanism based on the synthesis of stress proteins or heat-shock
proteins (HSPs) and the enzyme trehalose-6-phosphate synthase, which are
linked and possibly act together to achieve greater survivability to stress
caused by hydrostatic pressure.  C 2013 Wiley Periodicals, Inc.

Keywords: hydrostatic pressure; larvae; Anastrepha ludens; stress proteins

INTRODUCTION

In México and Central America, one of the most important plagues in fruits such as
mangoes and citrus is the Mexican fruit fly Anastrepha ludens. The main damage produced
by this fly is in the larval stage. This stage is subdivided in three development states, which
can last together between 5 and 8 days. Diverse methods of control have been developed
in order to counteract the economic losses caused by this insect. At present, larvae control
is achieved through programs of integrated handling of plagues that are applied at
regional and national level for the establishment of plague-free areas (Reyes et al., 2000;
Enkerlin, 2005). The most common methods of control in these programs have been the
application of toxic baits (e.g., hydroxylated malathion protein) and the release of sterile
insects (Knipling, 1953; Norrbom and Kim, 1988). Biological control of pests using only
physical methods other than hydrothermal treatments has been insufficiently explored;
one of such methods is high hydrostatic pressure (HHP). HHP is a nonthermal process
that consists in subjecting foodstuffs to high pressure through a fluid; the transmitting
fluid of the pressure is usually water, which is the origin of the term HHP (Hoover,
1997). This pressure is transmitted in a uniform and instantaneous way, independent
of the size, geometrical form or composition of the foodstuff (Cheftel, 1991; Parker
and Mehta, 2007). Velazquez et al. (2010b) and Candelario et al. (2010) evaluated the
ability of HHP (25–150 MPa) in combination with heat treatment (50◦ C) to inactivate
eggs and larvae of A. ludens. The authors reported that the combination treatment was
effective to inactivate both larvae and eggs to 150 MPa, but below this pressure a large
percentage of eggs and larvae were able to produce adult flies after treatment. Velazquez
et al. (2010a) evaluated the combination of HHP (25–150 MPa) at low temperature
(0◦ C) to inactivate eggs and larvae of A. ludens, and found that below 150 MPa both
eggs and larvae were capable of generating adult insects. Candelario-Rodrı́guez et al.
(2009) studied the efficacy of HHP treatments against eggs and larvae of A. ludens and
the physiological response of Tommy Atkins mangoes. Castañon-Rodrguez et al. (2011)

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evaluated the potential of HHP to inactivate eggs and larvae of A. ludens in Ataulfo
mangoes. These two authors performed investigations in order to establish that the HHP
affects the survival and the development ability of the insect until the adult state. However,
in all methods of control against A. ludens, there are small numbers of insects that survive
the applied treatment, because organisms develop defense mechanisms against harmful
effects that alter their vital functions. One of these mechanisms can be associated to the
overexpression of a group of genes that code for a family of proteins known as stress
proteins (SPs). The heat-shock proteins (HSPs), a kind of SP, are a group of intracellular
polypeptides highly conserved throughout the evolution; HSPs play a protective role like
molecular chaperons in the cells in order to facilitate folding, intracellular transport,
or assemble and degradation of other proteins (Lindquist, 1986; Lindquist and Craig,
1988; Morimoto, 1993). These SPs are inactive in cells under nonstress environmental
conditions; however, they become activated in the cell as a response to a stress stimulus
(Wu, 1995). HSPs are grouped in families based on the homology of their sequence and
molecular weight (MW). These proteins can be found in the main cellular compartment
(Benjamin and McMillan, 1998; Coronato et al., 1999).
In this work, we report the analysis of three proteins produced by A. ludens as a
response to the stress caused by HHP. These proteins belong to the group of HSP, and
could be associated to the mechanism developed in larvae as a response to this type of
control method.

MATERIALS AND METHODS

Anastrepha Ludens Larvae

Fruit fly larvae from the Moscafrut plant in Metapa de Dominguez, Chiapas, México were
used in two stages: 5 days after hatching—L2 (second stage) and 8 days after hatching—L3
(third stage). Treatments for each larval stage were performed in triplicate at four pressure
levels: 50, 75, 100, and 150 MPa; each level was applied for four different exposure times
(0, 5, 10, and 20 min). Larvae from each larval stage to which no treatments were applied
were used as control groups.

Pressure Application

The hydrostatic high-pressure protocol was applied in a cold isostatic press CIP42260
(Avure Autoclave Systems, Columbus, OH) ranging from 0.1 to 200 MPa. The pressurizing
medium was a mixture consisting of 5:1 ratio of lubricant:water (120-B Hydrolubric, EF
Houghton and Co., Valley Forge, PA). The treatments were carried out at 25◦ C and the
time required to reach the desired pressure level ranged between 2 and 5 min.

Survival Rate

After the pressure treatments surviving larvae count was performed, and the survival rate
was estimated by the following formula:
 
Total larvae − Live larvae
× 100
Total larvae

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Protein Extraction

Protein was recovered using a bio-homogenizer M133/1281-0 (Biospec Products, Inc.,

Bartlesville, OK) set in high speed. Larvae were homogenized in a 1:1 ratio (w/v) in 50
mM phosphate buffer pH 7.0 (Ausubel et al., 1992), which was previously chilled at 4◦ C.
The larvae suspension was centrifuged in an Eppendorf microcentrifuge 5415R at 16,000
× g for 10 min at 4◦ C. The supernatant containing the soluble proteins was recovered,
frozen and kept at −70◦ C until used.

Protein Concentration

Was determined by the method of Bradford (1976).

Electrophoresis

One-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed

according to Laemmli (1970). In all cases, a 4% stacking gel and a 12% resolving gel
were used. Electrophoresis was performed at 20 mA. An aliquot of MW standard mixture
(SDS6H2; Sigma Chemical, St. Louis, MO) was included in all gels. All the samples were
normalized previously by protein concentration in order to load the same quantity of
protein on each well.

Western Blotting

A monoclonal antibody Anti-Heat-Shock Protein 70 (HSP70; Clone BRM-22) Mouse As-

cites Fluid Product No. H 5147 (Sigma Chemical) was employed. Electrophoresis was
made as described previously. The transfer was performed using a similar condition
Trans-Blot SD Semidry Electrophoretic Transfer Cell #170–3940 (BIO-RAD, Hercules,
CA), as specified by the Instruction Manual, catalog number 170–3940 (BIO-RAD). For
the preparation of the required buffer, we followed the methodology for BIO-RAD. The
membrane was blocked with skim milk, washed with transfer buffer, and the blotting was
incubated in Grade Affinity Purified Goat Anti-Rabbit IgG (H + L). Alkaline Phosphatase
Conjugate, catalog number 170–6518 (BIO-RAD) was used for developing the Alkaline
Phosphatase Conjugate Substrate Kit, catalog number 170–6432 (BIO-RAD).

Densitometry of SDS-PAGE

SDS-PAGE gel was subjected to densitometry analysis with the Image Lab software version
4.0 in the molecular imager Gel Doc XR+ (BIO-RAD).

Protein Identification

The bands of interest were excised from the SDS-PAGE, samples were digested “in gel”
with trypsin and the resulting peptides were injected to a LC-MS (liquid chromatograph-
mass spectrometer) consisting of a microflow Accela (Thermo-Fisher Co., San Jose, CA)
with “splitter” (1/20) and a mass spectrometer LTQ-Orbitrap XL (Thermo-Fisher Co.)
with a nanotype electrospray ionization (ESI) system. For the fragmentation of the pep-
tides using the methods of CID (collision-induced dissociation) and HCD (high-energy
collision dissociation). Only charged ions 2+ and 3+ were selected for fragmentation

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Table 1. Percent Survival (±Standard Deviation) of A. ludens Larvae Subjected to High Hydrostatic
Pressures
Pressure (MPa) Time (min) Percentage of survival L2 Percentage of survival L3

50 0 8.3 ± 1.2efgh 11.5 ± 1.1def

5 1.6 ± 0.7j 21.4 ± 0.9a
10 4.3 ± 4.0ghij 8.9 ± 5.4efg
20 3.3 ± 2.6hij 17.3 ± 8.1abc
75 0 4.3 ± 1.7ghij 15.4 ± 5.2bcd
5 3.4 ± 2hij 12.6 ± 1cde
10 0.7 ± 0.4j 4.5 ± 0.8ghij
20 1 ± 0.1j 2.3 ± 2j
100 0 7.4 ± 0.5fghi 18.3 ± 7.1ab
5 0.1 ± 0.2j 9.4 ± 5.8efg
10 0.2 ± 0.1j 2.4 ± 0.3ij
20 0.1 ± 0.1j 0.3 ± 0.1j
150 0 0.7 ± 0.1j 3.4 ± 0.3hij
5 0.1 ± 0.1j 0 ± 0j
10 0.2 ± 0.1j 0 ± 0j
20 0 ± 0j 0 ± 0j

Treatments with the same letter are not significantly different.

events. Charged ions 1+ , greater than 4+ and indefinite were not considered. All spectra
were acquired in positive detection mode. During the automatic capture of data, dynamic
exclusion of ions was used, with an exclusion time of 60 sec. Spectrometric data were
subjected to search against the database NBCInr (All) through the Protein Prospector
program.

Statistical Analysis

Data were subjected to analysis of variance with the general linear model (GLM) using
SAS (Statistical Analysis System v. 8.0); the means were compared by the Duncan test using
the same software.

RESULTS

Index of Survival

The index of survival of between the treatments L3 and L2 showed differences at P <
0.001, L3 had greater index of survival than L2 at pressure levels of 75 and 50 MPa,
whereas for levels of 150 and 100 MPa there were no significant differences (P ≥ 0.05)
in the index of survival between larval stages. This is probably because at the lower
levels of applied pressure larvae from the third stage developed a better mechanism of
resistance; nevertheless, from the 100 MPa and up, the pressure effect was rather severe,
and produced larval death irrespective of their stage of development. The capacity of the
larvae to survive the treatments decreased as the level of pressure increased (Table 1).
These results agree with the report by Neven et al. (2007) who applied pressures between
0.48 and 2.15 MPa on the three different larval stages of the cherry western fly (Rhagoletis
indifferens). These authors found that when pressure increased, the level of mortality also
increased. Similarly, they also found that the third larval stage was the most resistant to

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Figure 1. Protein concentration in each treatment (μg protein/μl extract).

the treatments. Thomas and Shellie (2000) reported an increase in thermotolerance of

larvae from 10-day-old A. ludens (third larval stage), which provides more data supporting
greater resistance for the third larval stage of A. ludens.

Protein Concentration

The analysis of protein concentration in the extracts of larvae by Bradford revealed a

change in the protein content of the pressurized larvae, since significant differences
were found (P < 0.001) between the control and the treated groups, which was similar
between treatments with identical levels and times of pressure but in different stages of
development, where also significant differences were noted (P < 0.001). The highest
values of micrograms of protein by each microliter of extract were in the larvae of third
stage; although a noticeable difference between different stages of larval development
existed, significant differences between treatments with the same state of development
were not found (Fig. 1 and Table 2).

SDS-PAGE

No significant changes were found in the electrophoretic patterns of L2 protein extracts

(Fig. 2). However, electrophoresis for L3 extracts showed an increase in the amount of
protein of three specific bands (Fig. 3). The first band is observed at the top of the gels
with an MW near 70 kDa, in the third stage of the control group (L3). There is also a
constitutively increased presence of these proteins with MWs compared with the control
group L2. The band is presented in the middle of the gels with proteins having a MW of
ca. 30 kDa; the third band is shown in the lower region and contains proteins with MWs
near 23 kDa. These bands are not considered as constitutive in the control groups.

Western Blotting

Analysis of Western blotting confirmed the presence of HSPC of 70 kDa in larvae of

A. ludens of the third stage (L3) and in the control group (Fig. 4J) in which there is

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Table 2. Protein Concentration (±Standard Deviation) in Pressurized Larvae and Controls (μg Protein/μl
Extract)

Treatment Time (min) L2 (μg protein/μl extract) L3 (μg protein/μl extract)

50 MPa 0 2.17 ± 0.06gh 3.77 ± 0.09cde

5 2.30 ± 0.10h 3.87 ± 0.07bcd
10 2.23 ± 0.15gh 4.14 ± 0.06ab
20 2.13 ± 0.14h 3.96 ± 0.08bcd
75 MPa 0 2.30 ± 0.11gh 3.95 ± 0.13bcd
5 2.14 ± 0.13h 4.06 ± 0.08abcd
10 2.33 ± 0.21fgh 3.76 ± 0.11cde
20 2.50 ± 0.12fg 3.87 ± 0.07bcd
100 MPa 0 2.20 ± .20gh 3.91 ± 0.53bcd
5 2.10 ± 0.17h 3.72 ± 0.12de
10 2.37 ± 0.15fgh 3.98 ± 0.10bcd
20 2.60 ± 0.12f 4.35 ± 0.50a
150 MPa 0 2.32 ± 0.10fgh 3.97 ± 0.08bcd
5 2.42 ± 0.11fgh 3.94 ± 0.13bcd
10 2.35 ± 0.13fgh 3.94 ± 0.08bcd
20 2.23 ± 0.12gh 4.11 ± 0.13abc
Control L2 1.80 ± 0.10i
Control L3 3.51 ± 0.14e

Treatments with the same letter are not significantly different.

constitutive expression of these proteins. Similarly, this technique allowed us to observe

an increase in the content of these proteins in the larvae subjected to the different levels
of pressure (Fig. 4B–I).

Protein Identification

Identification of the overexpressed protein bands (Fig. 3) revealed that the bands with an
MW of 31 kDa were fully identified as trehalose-6-phosphate synthase (TPS1). As shown
in Figure 5, whereas a band with a MW of 23 kDa probably corresponds to fragments of
proteins having the highly conserved amino acid chain that belong to constitutive heat
shock proteins. As shown in Figure 6, the same that has been reported for Sarcophaga
crassipalpis. The results of samples 2 and 3 are reported as belonging to other genera of
different flies of A. ludens, since no genome or protein sequences specifically are reported
for the species A. ludens.

Densitometry Analysis

Densitometry applied to SDS-PAGE (Figs. 7 and 8) suggests that there is an increase in the
intensity of the bands of the first zone in the samples of pressurized larvae as the control
group L3 presented values of 3,466 and 3,609 lie that the treatments had values greater
than 3,568 (for treatments of 50 and 75 MPa) and 3,668 (for treatments of 100 and 150
MPa); this suggests an overexpression of proteins in this area. For area 2, the control
group had a value of 1 while for all pressure treatments larvae showed values above 1,022,
which implies that trehalose 6-phosphate synthase is constitutively present; however, and
after subjecting the larvae to pressure, this enzyme was produced in greater quantities.
For zone 3, the results suggest that SPs of 23 kDa are not expressed constitutively in the
control group; however, in extracts of pressurized larvae the presence of these proteins

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 Stress Proteins in Fruit Fly Larvae r 203

Figure 2. SDS-PAGE of A. ludens in second stage, control (L2) and treatment.

can be noted, which leads us to consider a possible de novo synthesis of these proteins as
a stress response caused by the HHP.

DISCUSSION

Figures 1 and 3 depict an increment in the protein content in L3. This increase can be
attributed to the synthesis of SPs as a measure of the larvae to resist damage caused by
the HHP. Organisms react against certain environmental stresses with a cellular defense
mechanism involving overexpression of these proteins and the induction of others of the
same family that are not constitutive. Their function is to minimize the damage caused by
stress (Ritossa, 1962). For the upper range of MW, proteins can be inferred to belong to
the HSP70 family, which includes proteins with MWs of 68–78 kDa; for middle and lower
parts, with the superfamily of small SPs includes proteins with MWs between 13 and 35 kDa
(Hass, 1991; Leppa and Sistonen, 1997). The HSP group, that do not constitute part of
the cell, but which are inducible as a response to stress, performs functions ranging from

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Figure 3. SDS-PAGE of A. ludens in third stage, control (L3) and treatment. The arrows indicate the increase
or the appearance of new bands in the electrophoretic pattern of larvae pressurized.

the removal of denatured proteins to increase the production of other proteins required
by the cell (Morimoto et al., 1990). It is possible to relate the overexpression of proteins
in the third larval stage (L3) with its increased ability to survive the elevated pressure,
but instead, the second larval stage (L2) had a smaller protein content in both the basal
control group and in the pressurized larvae. The difference between the basal levels of the

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Figure 4. Identification of HSP70 by Western blotting in A. Ludens, pressurized and control group (L3). (A)
Molecular weight marker; (B) 150 MPa, 20 min; (C) 150 MPa, 0 min; (D) 100 MPa, 20 min; (E) 100 MPa 0 min;
(F) 75 MPa, 20 min; (G) 75 MPa, 0 min; (H) 50 MPa, 20 min; (I) 50 MPa, 0 min; (J) control L3.

Figure 5. Alignment of the peptides obtained of the analysis of trypsin digestion, gray areas. Those pep-
tides share homology with several trehalose-6-phosphate synthase enzymes reported for different flies varieties.
Glossina morsitans morsitans, accession ADD20329; Drosophila sechellia, accession XP_002036065.1; Drosophila erecta,
accession XP_001970152.1; Drosophila melanogaster, accession NP_609114.1; Drosophila pseudoobscura pseudoob-
scura, accession XP_001357440.2.

Figure 6. The 23 kDa protein was analyzed by trypsin digestion and the resulting peptide was aligned with
sequences reported for HSP. The peptide obtained from the digestion, gray areas, share homology with several
heat shock proteins reported for different fly varieties. Lucilia cuprina, accession AEF38375.1; Sarcophaga cras-
sipalpis, accession AAC63387.1; Glossina morsitans morsitans, accession ADD18976.1; Ceratitis capitata, accession
ACG58884.1; Bactrocera dorsalis, accession AEJ88464.1.

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Figure 7. Densitometry of SDS-PAGE to L3 (50 and 75 MPa). The increase in the amount of protein is reflected
in the intensity of the coloration of the bands.

two larval stages can be attributed to some growth factors also generate slight increases
in the production of SPs and this process is fully independent and to a lesser extent than
those proteins induced by stress mechanisms. Factors that increase the production of con-
stitutive HSP are mitosis, growth factors, and cell differentiation processes (Welch, 1993;
Kindas-Mgge and Trautinger, 1994; Trautinger et al., 1995). Visual inspection of Figure 3
illustrates that in the control group L3 the expression of HSP70 is less than in pressurized
larvae. This is because the HSP represents between 2 and 15% of the total proteins in nor-
mal cells, but this proportion may reach 20% in cells exposed to stress (Donati et al., 1990;
Kopecek et al., 2001; Nollen and Morimoto, 2002). Tanguay (1983) affirmed that HSPs
are not necessarily synthesized de novo, but there are some that occur at baseline concen-
trations, and their synthesis is increased in response to some types of stress. Some HSPs
are constitutively expressed in the cytoplasm, endoplasmic reticulum, and mitochondria
to baseline levels in the absence of physiological stress (Anderson et al., 1994; Lund et al.,
1998; Perdue et al., 1998; Latchman, 2001). Moreover, a group of small molecules formed
by HSP of 15–30 kDa appears in large quantities with increasing temperature or other
conditions of stress (Jakob et al., 1993). Although the function of low-MW HSP as chaper-
ones is not clear, there is evidence suggesting that these HSPs stabilize the soluble proteins

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Figure 8. Densitometry of SDS-PAGE to L3 (100 and 150 MPa). The increase in the amount of protein is
reflected in the intensity of the coloration of the bands.

against denaturation by heat in vitro, prevent agglomeration of protein and promote the
proper reassembly of heat-denatured proteins (Yeh et al., 1994). Overexpression of HSP
in L3 can also be induced by denaturation of constitutive proteins of the cells, since it has
been reported that hydrostatic pressure causes damage to the structure and conforma-
tional changes of proteins. Changes in environmental conditions such as composition,
temperature, and pressure of the surrounding medium can disturb the proper balance of
intermolecular interactions of the protein and the surrounding medium and may lead to
the deployment and/or denaturation of the protein (Masson, 1992). Pressure can cause
a variety of effects on proteins, by inducing reversible or irreversible structural changes
that lead to protein denaturation, aggregation, or gel formation (Yaldagard et al., 2008).
It is reasonable to propose that denaturation occurs in cellular proteins and therefore
leads to overexpression of SPs, since the function of these proteins is precisely to main-
tain the stability and proper folding of the constitutive proteins necessary for proper cell

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metabolism. The intracellular accumulation of denatured or badly folded proteins as a

result of a stressful situation triggers a response that involves the production of high levels
of HSP, which facilitates the rearrangement of defective proteins. The rapid induction of
HSPs is due to the activation of a transcription factor (HSF1), which increases the activity
of genes encoding for these proteins (Morimoto et al., 1990). The plot of intermolecular
interactions that stabilizes the quaternary structure of oligomers is particularly sensitive to
pressure and acts as a driving force for the formation of aggregates and protein gelation
(Smeller et al., 1999). This is the perfect setting to trigger the primary function of HSPs,
which is reflected in the electrophoretic patterns (Fig. 3) of L3. The increase of HSP70 is
a response to the stressing effects of pressure, given its ability to be in the correct folding
of chaperones for proper three-dimensional conformation of proteins on ribosomes, the
refolding of denatured proteins after severe stress and auxiliary protein degradation in
severely damaged organisms (Kiang and Tsokas, 1998). In particular, HSP70 belongs to
the group of proteins known as molecular chaperones, which interact with a wide variety
of other cellular proteins to stabilize its conformation and prevent protein aggregation
or breakdown under stress; low-MW HSP also have this protective effect (Sabehat et al.,
1995). L3 shows the overexpression of proteins from two groups, HSP70 and the low-MW
HSP (Figs. 4–8). This makes them more resistant to stress. Some authors believe that there
is an effective cooperation between these two superfamilies in nature as we observed in
this study. Sabehat et al. (1995) used Western blotting in samples from preheated tomato
extracts and HSP70 accumulation was observed, as well as the occurrence of low-MW
HSP, in the range of 18–21 kDa, in addition to the appearance of proteins in the range of
38–42 kDa. Overexpression of members of the HSP70 family and small SPs in Drosophila
and Caenorhabditis elegans has shown to extend the life span of these organisms, which
have implicated heat-shock factors as regulators of longevity (Tatar et al., 1997; Yokoyama
et al., 2002; Walker and Lithgow, 2003). Yin (Yin et al., 2006), reported that accumulation
of HSP70 family proteins together with low-MW stress confer thermotolerance in larvae
of Cydia pomonella (Lepidoptera: Tortricidae). The results agree with previous reports in
different organisms, as for protein overexpression of two superfamilies: the HSP70 and
low-MW HSP. The phenomenon of barotolerance in living cells is not fully understood,
but appears to involve an interaction between trehalose and SPs (Iwahashi et al., 1998;
Fernandes, 2005; Purvis et al., 2005). The combination of molecular structure and physic-
ochemical properties of trehalose make it a very stable disaccharide (Birch, 1963; Elbein,
1974). It has been reported that trehalose may be involved in supporting, regulation, or
exercise functions to protect membranes and proteins against the adverse effects of stress
caused by heat, cold, desiccation, and anoxia (Crowe et al., 1984). Trehalose is a disac-
charide produced in large quantities by various organisms in response to various stress
agents. Trehalose prevents protein denaturation at high temperatures in vitro, but its role
in stress tolerance in vivo is still controversial (Singer and Lindquist, 1998). This suggests
that the presence of trehalose during stress is important, which in turn requires a large
amount of the disaccharide to promote protein and cell membrane stability, and justifies
the overexpression of proteins; in this case an enzymatic function related to the synthesis
of trehalose. As discussed above, the results of this study suggest in Figure 3 for sample
2, the overexpression of the enzyme trehalose-6-phosphate synthase (TPS1). TPS1 is the
first enzyme in the biosynthetic pathway of trehalose and is responsible for converting
glucose to trehalose-6-phosphate (Bell et al., 1992). Soto et al. (1999) proposed that the
accumulation of trehalose synthesized by the trehalose-6-phosphate synthase is the first
mechanism of resistance to stress. Conversely, Singer and Lindquist (1998) argue that
the role of trehalose to protect proteins in the initial stages of the stress response before

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HSPs has been fully induced. This would give an alternative explanation for a cross talk
between trehalose synthesis and the synthesis of HSP in response to stress (Soto et al.,
1999).

CONCLUSIONS

Larvae of A. ludens show a biochemical response to different treatments under pressure,

which depends on their state of development. Third instar larvae (L3) developed a better
protection mechanism based on the overexpression of HSP70 SPs, the enzyme trehalose-
6-phosphate synthase, production of trehalose and SPs of 23 kDa, which act together to
provide greater capacity to survive the stress caused by hydrostatic pressure. The results
of this study suggest that both the HSP overexpression as synergistic TPS1 have clear roles
in the response to stress, since by increasing the trehalose-6-phosphate synthase trehalose
content was increased, which can serve as protein stabilizer while the HSP synthesis of 23
kDa protein is performed.

ACKNOWLEDGMENTS

Financial support of PROMEP (Mexico) through a network grant for utilization of agri-
cultural resources is gratefully acknowledged.
The authors declare no conflicts of interest.

LITERATURE CITED

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