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The importance of starch and sucrose digestion in nutritive biology of synanthropic acaridid mites╬▒-Amylases and ╬▒-glucosidases are suitable targets for inhibitor-based strategies of mite control.

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A r t i c l e
THE IMPORTANCE OF STARCH AND
SUCROSE DIGESTION IN NUTRITIVE
BIOLOGY OF SYNANTHROPIC
ACARIDID MITES: a-AMYLASES
AND a-GLUCOSIDASES ARE
SUITABLE TARGETS FOR INHIBITORBASED STRATEGIES OF MITE
CONTROL
Tomas Erban, Michaela Erbanova, Marta Nesvorna, and
Jan Hubert
Crop Research Institute, Prague, Czech Republic
The adaptation of nine species of mites that infest stored products for
starch utilization was tested by (1) enzymatic analysis using feces and
whole mite extracts, (2) biotests, and (3) inhibition experiments. Acarus
siro, Aleuroglyphus ovatus, and Tyroborus lini were associated with the
starch-type substrates and maltose, with higher enzymatic activities
observed in whole mite extracts. Lepidoglyphus destructor was associated
with the same substrates but had higher activities in feces. Dermatophagoides farinae, Chortoglyphus arcuatus, and Caloglyphus redickorzevi
were associated with sucrose. Tyrophagus putrescentiae and Carpoglyphus lactis had low or intermediate enzymatic activity on the tested
substrates. Biotests on starch additive diets showed accelerated growth of
species associated with the starch-type substrates. The inhibitor acarbose
suppressed starch hydrolysis and growth of the mites. We suggest that the
species with higher starch hydrolytic activity in feces were more tolerant to
acarbose, and a-amylase and a-glucosidase of synanthropic mites are
Grant sponsor: Czech Science Foundation; Grant number: GACR525/07/P253; Grant sponsor: Ministry of
Agriculture of the Czech Republic; Grant number: MZE0002700604.
Abbreviations: A, amylopectin; Acarbose, 400 ,600 -Dideoxy-400 ([1S]-[1,4,6/5]-4,5,6-trihydroxy-3-hydroxymethyl-2yclohexenylamino)-maltotriose; BR-I, Britton-Robinson I buffer; Glc, glucose; IgE, immunoglobulin E; M,
maltose; RH, relative humidity; SGME, feces extract; WME, whole mite extract; WS, wheat starch
Correspondence to: Tomas Erban, Crop Research Institute, Drnovska 507, Praha 6-Ruzyne, Czech Republic
CZ 16106. E-mail: arachnid@centrum.cz
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 71, No. 3, 139–158 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20312
140
Archives of Insect Biochemistry and Physiology, July 2009
C 2009
suitable targets for inhibitor-based strategies of mite control. Wiley Periodicals, Inc.
Keywords: a-amylase; a-glucosidase; a-amylase/a-glucosidase inhibitor;
synanthropic acaridid mite; digestion
INTRODUCTION
It has been suggested that synanthropic mites (Acari: Acaridida) enter into human
homes, stored foods, and animal feeds from the soil through bird and rodent nests
(OConnor, 1979). For this type of successful colonization, physiological preadaptations have been predicted. In mite-feeding biology, the appropriate digestive
enzymes have been suggested to be the key for nutrient utilization and successful
colonization of various habitats (Luxton, 1972; Bowman and Childs, 1982; Bowman,
1984; Siepel and de Ruiter-Dijkman, 1993).
Generally, carbohydrates (saccharides) are the most abundant class of organic
compounds found in stored plant products, especially in cereals. In addition,
carbohydrates are an easily obtained and readily digested form of fuel in nature,
and in human diets starch and sucrose are the main digestible carbohydrates (Nichols
et al., 2003). The enzymatic machinery used to digest common carbohydrates, such as
starch and sucrose in grains, is ubiquitous among living organisms. Starch is degraded
by a combined action of amylases, a-glucosidases, and a-dextrinases. The products of
a-amylase are maltotriose and maltose from amylose. Amylopectin is hydrolyzed to
maltose, glucose, and limit dextrin. The sucrose is degraded by invertase (a-Dglucosidase) (Voet and Voet, 2004).
The enzymatic activities of a-amylases and a-D-glucosidases have been observed in
many species of mites (Bowman, 1984; Bowman and Lessiter, 1985; Akimov, 1985;
Morgan and Arlian, 2006). However, differences in the a-amylases and a-glucosidases
activities among synanthropic mite species, which indicate different adaptations to
starch digestion, have not been significantly reported in the literature.
To determine the potential importance of starches and sucrose in the nutritive
biology of synanthropic mites, we developed a new approach. In this study, we applied
(1) enzymatic analysis, (2) biotests, and (3) inhibition experiments to analyze nine
species of synanthropic mites. The comparison of amylolytic and a-glucosidase
activities among the tested species was based on a comparison of the amounts of
glucose liberated from the starches, starch-type substrates (dextrin, amylopectin, and
maltose), and sucrose after hydrolysis by the extracts from the whole mite extracts
(WME) and the spent growth medium extracts (SGME).
MATERIALS AND METHODS
Experimental Mites
Nine species of stored-product mites and one house-dust mite, all of medical and
economic importance (Hughes, 1976), were selected for this study. Five families of
acaridid mites were studied: Acaridae: Acarus siro (Linnaeus, 1758); Aleuroglyphus
ovatus (Troupeau, 1878); Caloglyphus redickorzevi (Zachvatkin, 1941) [syn. Caloglyphus
Archives of Insect Biochemistry and Physiology
Digestion of Starch Substrates and Sucrose by Mites
141
hughesi (Samsinak, 1966)]; Tyrophagus putrescentiae (Schrank, 1781); Tyroborus lini
(Oudemans, 1924); Carpoglyphidae: Carpoglyphus lactis (Linnaeus, 1758); Chortoglyphidae: Chortoglyphus arcuatus (Troupeau, 1879); Pyroglyphidae: Dermatophagoides
farinae (Hughes, 1961); and Glycyphagidae: Lepidoglyphus destructor (Schrank, 1871).
The analyzed species originated from laboratory stock cultures kept in the Crop
Research Institute, Prague. The mites were mass-reared on the wheat and fish foodderived rearing diet as described in Erban and Hubert (2008). Based on preliminary
observations, the diet itself was free of any amylolytic and a-glucosidase (maltase and
saccharase) activities.
Preparation of the Samples for Biochemical Analyses
The enzymatic assays were performed with two different extracts: (1) whole mite
extracts (WME) and (2) the spent growth medium extracts (SGME). As the source of
feces, we used the contents of the rearing chambers after separation from the food by
sieving with a Retsch sieve (0.25 mm) using an AS 200 digital analytical sieve machine
(Retsch, Haan, Germany). The material was collected at 10-day intervals. The
fragments of food and eggs were removed manually using a STEMI 2000 dissection
microscope (C. Zeiss, Jena, Germany). The collected material was stored in a deep
freezer at 401C. The samples were extracted in a physiological solution of 0.9% (w/w)
NaCl in ddH2O (Cat. No. 95304, Sigma-Aldrich, Saint Louis, MO) in an ice bath. The
feces were transferred into sterilized 25-mL Erlenmeyer glass flasks. To 0.1 g of
material, 2 mL of a cold physiological solution of 0.9% (w/w) NaCl in ddH2O was
added. The flasks were covered with parafilm and shaken on a PST-60 HL Plus
temperature-controlled shaker (Biosan, Riga, Latvia) at 470.51C using a ES 500
thermostat (Nüve, Ankara, Turkey) for 12 h. The extract was collected with a
micropipette.
To obtain the WME, the mites were first collected from the rearing chambers using
a brush-pencil under a dissection microscope at 20-day intervals. The mites were
transferred to a 20-mL plastic syringe (Chirana T. Injecta a.s., Stara Tura, Slovakia)
that was modified by cutting the front side and coated with a double layer of muslin.
The mites were cleaned in a 0.1% (w/w) solution of detergent Triton X-100 (Serva
Electrophoresis GmbH, Heidelberg, Germany) in ddH2O, and the procedure was
repeated five times. Washing in ddH2O removed the remnants of the detergent. The
cleaned mites were transferred into a glass Potter-Elvehjem homogenizer (Kavalier,
Sazava, Czech Republic) and were homogenized in the physiological solution in an ice
bath.
Both WME and SGME homogenates were centrifuged in a MR 23i centrifuge
(Jouan Industries S.A.S., France) at 41C for 5 min at 13,500g. The supernatant was
transferred into new Eppendorf tubes and centrifuged for 10 min at 33,000g. Finally,
the protein content was determined using the Bradford reagent (Cat. No. B6916,
Sigma-Aldrich) at 595 nm (Multiskan Ascent, Thermo, Shanghai, China). A protein
standard (Cat. No. P0834, Sigma-Aldrich) was used for the calibration. Consequently,
the extracts were diluted to a concentration of 500 mg/ml for WME and 100 mg/mL for
SGME and stored in Cryovials (Cryovial, Beloeil, Canada) at 401C.
Determination of pH Optima of Starch Digestion and a-Glucosidases
Wheat starch (Cat. No. S2760, Sigma-Aldrich) was used as the substrate for screening
of pH optima for starch digestion of two selected model species, Acarus siro and
Archives of Insect Biochemistry and Physiology
142
Archives of Insect Biochemistry and Physiology, July 2009
Aleuroglyphus ovatus. The wheat starch substrate was prepared at a concentration of
0.1 g in 0.125 mol/L Britton-Robinson I (BR-I) buffer with 0.5 mol/L CaCl2 in a wide
range of pH values (2.0–10.0); the final concentration of BR-I in the reaction was
0.1 mol/L. With continuous stirring using a disperser (Ultra Turraxs T8, IKAsWERKE GmbH a Co. KG, Staufen, Germany), 200 ml of fresh prepared starch
suspension was aliquoted into each well of 300 ml microplates (GAMA a.s., Ceske
Budejovice, Czech Republic). The microplates were covered with plastic lids and
preincubated for 10 min at 371C in a thermo shaker (DTS-2 ELMI Ltd., Riga, Latvia).
The assay was initiated by the application of 50 ml of homogenate sample using a
multichannel pipette. Six replicates and two blanks were performed per pH. The
microplates were covered with adhesive sealing film for microplates (Simport, Beloeil,
Canada) and the assay was incubated for 1 h for WME and for 2 h for SGME at 371C
and 900 rpm in the thermo shaker (DTS-2). The reaction was stopped by addition of
30 ml of 3 M trichloroacetic acid after removal of the sealing film. The microplates were
then centrifuged for 10 min at 21C and 2,500 rpm in a MR 23i centrifuge (Jouan
Industries S.A.S., France). The glucose content was determined as described in
Determination of Glucose Content in the Reactions.
For the pH optima screening for maltase and saccharase, an assay analogous to the
determination of starch digestion was applied, but it was not stirred by a disperser
during the application of the substrate. The substrates used were sucrose (Cat. No.
S7903, Sigma-Aldrich) and maltose (Cat. No. M5885, Sigma-Aldrich), both in final
concentrations of 30 mmol/L in the reactions.
Comparison of Digestion of Starch-Type Substrates and Disaccharides
The ability to digest different types of starches, including amylopectin and dextrin,
and disaccharides, like maltose and sucrose, was tested on the appropriate commercial
substrates with the nine species of mites. The following substrates (all from SigmaAldrich) were applied in the enzymatic tests: starch from wheat (Cat. No. S7260),
starch from potato (Cat. No. S2630), starch from rice (Cat. No. 85564), starch from
corn (Cat. No. S4180), amylopectin (Cat. No. A8515), dextrin (Cat. No. D2006),
sucrose (Cat. No. S7903), and maltose (Cat. No. M5885). Although the selected
starches are from different sources, the most important differences are a result of the
purification processes (for details, see the manufacturer’s descriptions). All of the
substrates were prepared just before the test without heating. The assay was
performed as described above, except that the pH of the BR-I buffer in all assays
was 5.0 and the final molarity in the reaction was 0.1 mol.dm3.
The hydrolytic activity of both extracts was given as the concentration of glucose
(for details, see Determination of Glucose Content in the Reactions). The concentration data showed a normal distribution. ANOVA (Analysis of variance) tests were
applied separately for each substrate and type of extract and the species was the factor.
The Turkey’s HSD intervals enabled a comparison of differences among the species
at the P 5 0.05 probability level. The Pearson’s correlations were calculated to describe
the similarities among the enzymes or relationships between WME and SGME. The
replicates per species were randomized and the hydrolytic activities were compared.
Finally, the principal component analysis was calculated. In both analyses, the data
were the amounts of glucose obtained, the substrates extracts were the column
variable, and lines were the species replicates. All analyses were done in the XLSTAT
software (Addinsoft Inc, New York, NY).
Archives of Insect Biochemistry and Physiology
Digestion of Starch Substrates and Sucrose by Mites
143
In Vitro Inhibition of Starch, Amylopectin, and Maltose Hydrolysis in WME
A kinetic study was performed in an assay similar to that described above, but with a
modification based on the addition of the free enzyme form at the start of the reaction.
This modification enables us to observe the real competition between inhibitor and
substrate and is recommended for kinetic studies of competitive inhibitors (Copeland,
2000). The a-amylase and a-glucosidase inhibitor acarbose from Actinoplanes spp.
(Cat. No. 10017, Serva Electrophoresis GmbH, Heidelberg, Germany) was tested for
in vitro inhibition of starch, amylopectin, and maltose digestion. The substrate
solutions were prepared from wheat starch (Cat. No. S7260), maltose (Cat. No.
M5885), and amylopectin (Cat. No. A8515), and the final concentrations of those
substrates were the same as described above. The assay was performed with six
concentrations of acarbose and one control lacking acarbose, all in three replicates.
Serial dilutions were made starting from a concentration of 200 nmol/mL of acarbose.
The concentrations of inhibitor were selected according to the total activity on the
appropriate substrates. Ten microliters of acarbose was applied into the appropriate
wells of the microplate and 10 ml of BR-I without acarbose was used as a control. Then,
190 ml of substrate solution was added to each well and the microplates were
preincubated for 10 min at 371C. The assay was started by addition of 50 ml of WME,
each with a protein concentration of 500 mg/mL. The activity was expressed as the
relative percentage of remaining activity (vi/v0 102). The data were electronically
transferred to Origin 6.1s (OriginLab Corporation, Northampton, MA) and fitted by
exponential functions. The IC50 values were determined from the fits of the data at
50% of the remaining enzymatic activity.
Determination of Glucose Content in the Reactions
The glucose concentration was determined using the BIO-LA-TESTs GLUCOSE
GOD 1500 kit (Lachema-PLIVA, Brno, Czech Republic). The deproteinized solution
was transferred to a 96-well microplate and 200 ml of the GLUCOSE GOD 1500 kit was
added. The samples out of the calibration range were diluted. The absorbance was
read at 492 nm (lmax498 nm) after 30 min of incubation at 251C.
The homogenates were tested in the presence of glucose that originated from the
tissue or from hydrolysis of unknown carbohydrates before or during experiments. To
control for this, the WME and SGME were incubated in BR-I buffer but without the
substrates. The resulting concentration of glucose was determined from the total
concentration of glucose in the samples.
The data from in vitro analyses were electronically transferred to Origin 6.1s and
calibrated. The glucose content was determined from a standard curve, where glucose
was used as the standard (Cat. No. G8270, Sigma-Aldrich). The hydrolytic activities on
different substrates were expressed as the specific activity in mg/mL Glc h1 per mg/mL
of protein in the homogenate.
In Vivo Observation of Starch Digestion on the Gut Level
Starch azure (Cat. No. S7629; 0.1 mg) was served as a diet for at least 100 mites in
Eppendorf tubes. The mites that ingested the starch azure were collected after 24 h
under a dissection microscope, rinsed in a drop of physiological solution (0.9% NaCl),
and mounted onto wet plates on a physiological solution at 1:10 (v/v).
Archives of Insect Biochemistry and Physiology
144
Archives of Insect Biochemistry and Physiology, July 2009
The a-amylase activity liberated the covalently linked remazol brilliant blue
R (RBB) leading to a blue color. The blue color in the gut suggested the presence of
a-amylase activity. The elution of RBB was observed under a compound microscope
(Axioskop) using the Axiovison software (C. Zeiss, Jena, Germany). The minimal
design included five positive observations per species.
Biotests on Wheat Starch, Maltose, and Sucrose Additives in Diets
The experimental diets were derived from rearing diets (see above) and were enriched
by 5% (w/w) with starch from wheat, maltose, and sucrose, separately, in a modification
of a previously described biotest (Matsumoto, 1965). The carbohydrates were
dissolved in ddH2O and incorporated into the mite food; the calculated volume was
approximately 3 mL of solution per 1 g of food. The resulting combination was
properly mixed using a MS1 Minishaker (IKAs, Staufen, Germany) and lyophilized in
a PowerDry LL3000 (Thermo, Shanghai, China). The lyophilized material was ground
into a powder in a pottery grinding mortar. The material was rehydrated 24 h before
the experiment in a desiccator containing distilled water.
The unmodified rearing diet was used as the control. The diet (5 mg) and
10 adults were placed in tissue culture cell chambers (Cat. No. 3102025, IWAKI,
Saint Marcel, France). The chambers were incubated in desiccators at 75 or 85%
RH (see above) at 251C in the dark for 21 days (Erban and Hubert, 2008). The
experiment was terminated by the addition of 10 mL of 80% ethanol to the
chambers. The mites were directly counted under a Stemi 2000 C dissection
microscope. The experiment was usually designed with 12 replicates per diet and
species. As only the initial and final population densities were available, we expected
the population of mites to change according to a formula for exponential growth, and
a differential density-independent model (Nt 5 N0ert) was used to estimate the
observed rate of population increase (r), where N0 was the initial density of mites
(N0 5 10), Nt was the final density of mites, and t was the duration of the experiment
(21 days).
The rates of population increase showed normal distributions and were analyzed
by ANOVA. The factors were the substrates and analyses were done for each species
separately. Tukey’s (HSD) intervals were used for the comparison of differences among
substrates, including the control, at the P 5 0.05 probability level.
Biotest on the a-Amylase and a-Glucosidase Inhibitor
The inhibitor-containing diet was derived from the rearing diet with the incorporation
of acarbose (Cat. No. 10017, Serva Electrophoresis GmbH, Heidelberg, Germany).
Acarbose was incorporated in a range of 0.001–5 mg of acarbose per g of diet. The
unmodified rearing diet was used as the control. The diet (10 mg) and 50 adults were
placed in IWAKI tissue culture cell chambers with controlled conditions, i.e., 75 or
85% r. h. (see above) at 251C in the dark for 21 days. The rate of population increase
was checked by the same method as described above, except that N0 5 50. To
determine the effect of acarbose, the linear regression model was applied to the data.
The rate of increase was the independent variable, while the acarbose concentration,
species, and their interaction were the factors. In addition, the linear regressions were
done for all species separately to obtain the parameters of the regression line.
For orientation, the rc50 parameters were defined as the concentration of acarbose
Archives of Insect Biochemistry and Physiology
Digestion of Starch Substrates and Sucrose by Mites
145
when the population growth of the mites was 50% compared to the control (Hubert
et al., 2007).
RESULTS
Hydrolytic Activity of WME and SGME on Starch-Type Substrates and Disaccharides
The hydrolytic activities of both WME and SGME on starch-type substrates, including
wheat, potato, corn, and rice starches, amylopectin, dextrin, and disaccharides,
including maltose and sucrose, were observed in all species of mites (Table 1).
The hydrolysis of wheat starch, maltose, and sucrose substrates by the WME of A. siro
and A. ovatus showed similar maxima in 0.1 M BR-I buffer. All pH optima were between
pH 4 and 6.75, with a maximal activity at pH 5 on all substrates and both species. In
addition, the hydrolytic activity on sucrose displayed a second maxima at a strongly
acidic pH.
The ranks of the hydrolytic activities of the species were not the same in WME and
SGME on any substrate (Table 1). The most apparent difference occurred with
L. destructor, which had seven, eight, and four times higher hydrolytic activity on wheat
starch, amylopectin, and maltose in SGME than in WME, respectively. Similarly,
D. farinae had two times higher hydrolytic activity on wheat starch in SGME than in
WME, respectively. In spite of the differences between WME and SGME, the relative
data indicate that there were similar proportions of glucose obtained from the starches
in WME and SGME in all species (Table 1). The hydrolytic activity with sucrose was
completely different from starch and starch-type substrates as shown by the rank of the
species. The velocity of production of glucose and fructose from saccharose by WME
decreased in rank from D. farinae, C. arcuatus, C. lactis, T. lini, C. redickorzevi, A. siro,
A. ovatus, to T. putrescentiae. The hydrolytic activity of WME with sucrose was
significantly negatively correlated with the starch substrate and maltose. The negative
correlations were not significant in SGME.
The component analysis (PCA) compared the amount of glucose after hydrolysis
of starch substrates and sucrose by both mite extracts (WME or SGME). In the model,
the position of variables in the first two axes explained 74% of the total variance in the
dataset (Fig. 1). The variables position according to the first axis explained 48% of the
variability in the dataset. The position according to the first axis showed two separate
clusters; enzymatic activity against starch substrates (wheat, potato, corn, rice starches,
amylopectin, and dextrin) and the disaccharide maltose was separated from the
saccharolytic activity. The variables position according to the second axis explained
26% of the variability in the dataset. The position in the second axis distinguished
between the hydrolytic activities of WME and SGME. The species of mites were
distributed into four clusters according to their positions in the first two axes. The
mites in cluster (1), A. siro, A. ovatus, and T. lini, were associated with the type of starch
substrate and maltose hydrolysis, with high enzymatic activities in WME. Cluster (2)
contained only L. destructor and was associated with hydrolysis of the same substrates as
the previous species, but with the prevailing enzymatic activity in SGME. Cluster (3)
contained mite species associated with the saccharolytic activity, including D. farinae,
C. arcuatus, and C. redickorzevi. Cluster (4) consisted of species with low or intermediate
enzymatic activity on both the starch substrates and sucrose, i.e., T. putrescentiae and
C. lactis (Fig. 1).
Archives of Insect Biochemistry and Physiology
894
8.49
o0.001
ANOVA
F
d.f.
P
2.2770.06
2.0970.07
0.8770.03
0.4670.01
0.2470.02
0.2770.01
2.1270.05
2.0770.04
1.1170.06
2,715
8.49
o0.001
WME
A. ovatus
A. siro
C. arcuatus
C. lactis
C. redickorzevi
D. farinae
L. destructor
T. lini
T. putrescentiae
ANOVA
F
d.f.
P
a
b
d
e
f
f
b
b
c
T
cd
ef
de
fg
bc
b
a
de
g
T
100
100
100
100
100
100
100
100
100
d
e
bc
e
cd
bc
a
de
f
T
1,273
8.49
o0.001
2.3870.09
2.0170.04
0.8870.02
0.5570.02
0.2770.02
0.2470.01
2.1970.09
2.0770.04
1.1270.10
a
c
e
f
g
g
b
bc
d
T
Amylopectin
736
8.49
o0.001
2.2870.17
1.4070.09
3.1770.17
1.2170.07
2.6370.47
3.6170.28
17.1871.31
1.8770.20
0.3770.03
Mean7sd
% Mean7sd
100
100
100
100
100
100
100
100
100
%
105
96
101
122
111
87
104
100
101
%
91
96
171
141
94
103
110
92
99
%
2,147
8.48
o0.001
1.5470.04
1.3570.04
0.1770.004
0.0470.002
0.1970.02
0.2270.01
1.2170.03
1.2170.01
0.6570.03
Mean7sd
Potato
583
8.48
o0.001
1.3370.05
0.5570.06
0.3370.03
0.0870.06
1.2970.10
1.3370.02
2.1270.08
0.3970.02
0.1470.01
Mean7sd
Potato
53
38
18
9
46
38
14
19
37
Rice
123
8.49
o0.001
0.6470.02
0.2370.05
0.2070.05
0.0970.03
0.5570.08
0.427.0.05
0.4970.02
0.117.0.01
0.0470.003
Mean7sd
a
b
e
f
e
e
c
c
d
68
64
20
9
77
80
57
58
58
1,050
8.49
o0.001
0.9970.05
0.2270.02
0.1470.01
0.0370.01
0.0470.001
0.0570.01
0.4570.02
0.2770.02
0.1270.01
T % Mean7sd
b
c
d
e
b
b
a
d
e
T %
Rice
26
16
11
10
20
12
3
5
10
Corn
201
8.48
o0.001
0.5670.03
0.2870.03
0.1670.01
0.0570.01
0.4470.06
0.4270.02
0.4670.01
0.1070.01
0.0570.004
Mean7sd
a
c
d
e
e
e
b
c
d
44
11
16
6
18
20
21
13
11
734
8.48
o0.001
0.6670.05
0.1570.01
0.0670.005
0.0170.002
0.0270.003
0.0470.004
0.1870.008
0.1670.007
0.0970.004
T % Mean7sd
a
d
d
e
ab
c
b
e
e
T %
Corn
22
19
9
5
16
12
3
5
13
b
d
b
d
bc
b
a
c
e
T
1,377
8.45
o0.001
a 29 4.3570.10
b
7 3.5870.16
cd 7 1.2770.06
f
2 3.1870.11
ef 7 0.7470.03
de 16 0.4570.01
b
9 4.1770.13
b
8 3.7770.10
cd 8 2.067010
127
97
164
123
103
91
114
121
118
%
a
c
f
d
g
h
a
b
e
192
171
147
699
300
166
197
182
186
T %
Maltose
2,444
8.45
o0.001
3.2170.17
1.4270.05
3.0570.49
1.0570.07
2.8970.18
3.2070.15
17.8670.48
2.4670.19
0.4470.04
Mean7sd
T % Mean7sd
a
c
d
e
b
b
b
e
e
T %
Maltose
758
8.49
o0.001
1.9970.07
1.7170.06
0.7670.01
0.6570.02
0.4370.02
0.3570.02
1.9370.07
1.7370.11
1.0770.09
Mean7sd
%
Mean7sd
Mean7sd
1,246
8.45
o0.001
T
%
228
10
179
268
38
102
1
2
14
%
de
5
de
8
b
103
c
137
d
87
a 1455
de
4
c
28
e
3
Sucrose
a
88 0.1170.001
b
82 0.1670.003
d
87 0.9070.02
e 143 0.6270.06
f 176 0.2170.01
g 127 3.9770.25
a
91 0.0870.003
b
83 0.5870.01
c
97 0.0370.06
T %
443
8.45
o0.001
a
e
b
c
d
b
e
e
e
T
Sucrose
cd 92 5.7370.23
e
92 0.1570.03
cd 135 3.3270.56
f
77 2.3070.36
cd 80 1.0670.07
b
93 3.6070.08
a
33 0.2070.01
de 90 0.0570.03
f 115 0.0570.01
T
Dextrin
174
8.39
o0.001
2.3270.44
1.3470.16
2.5170.06
0.6670.07
2.2470.43
3.2770.24
5.1870.45
1.8370.22
0.4370.0.2
Mean7sd
Dextrin
The relative activity (%) is normalized to the activity on wheat starch. The data are means and standard deviations. The letters indicate the Turkey’s HSD groups at the p 5 0.05
significance level.
Mean7sd
Species
Wheat
2.5270.03
1.4670.05
1.8670.25
0.8670.13
2.8170.42
3.5270.11
15.6271.08
2.0370.04
0.3770.01
Mean7sd
Amylopectin
SGME
A. ovatus
A. siro
C. arcuatus
C. lactis
C. redickorzevi
D. farinae
L. destructor
T. lini
T. putrescentiae
Species
Wheat
Table 1. The Hydrolytic Activity of WME and SGME on the Tested Starch Substrates and Sucrose Expressed as Specific Activity (mg/mL Glc h1 per mg/mL of protein in homogenate)
146
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Digestion of Starch Substrates and Sucrose by Mites
147
Figure 1. Centroid biplot of principal component analyses. The data are the specific enzymatic activities
expressed as the amounts of glucose obtained (mg/mL Glc h1 per mg/mL of protein in homogenate), the
substrates extracts are the column variable, and the lines are species replicates. The species are expressed
as centroids and substrates extracts as the points. The position of the species and extracts according to two
first axes explained 74% of variability in the data set.
Observation of a-Amylase Activity in the Gut of Mites
The mites ingested starch azure as a diet. The ingested particles were concentrated in
the ventriculus (v) (Fig. 2A) and formed food boli (fb) (Fig. 2B) (for gut terminology,
see Sobotnik et al., 2008). The remazol brilliant blue (RBB) released after hydrolysis
showed the blue color mainly in the ventriculus and caeca (ca) (Fig. 2E). It was clearly
apparent in species with (Fig. 2F) and without food boli (Fig. 2C) in the ventriculus,
indicating that the immediate starch is hydrolyzed after starch azure ingestion. In
D. farinae, the separate particles of starch azure were observed in the ventriculus and
the blue color entered into granules (Fig. 2D). The food boli were passed from the
ventriculus to the postcolon (pc) and showed visible separation of the blue color from
the food boli in the postcolon (Fig. 2B, C, E). The hydrolysis of starch continued from
the ventriculus to the colon as shown in L. destructor. The RBB from food bolus in the
colon was released during 3 h in one specimen and the concentration of the blue color
decreased with increasing time of digestion (Fig. 2G).
On the interface of caecal cells and the postcolon, the RBB entered into the caecal
cells as indicated by the high concentration of RBB inside the cells (Fig. 2E).
The Effect of Wheat Starch, Maltose, and Sucrose Additives in the Rearing Diet on the
Population Growth of Acaridid Mites
T. putrescentiae, A. siro, T. lini, L. destructor, and A. ovatus showed more accelerated intrinsic
growth rates on a diet containing wheat starch than on the control diet (Table 2). No
differences or decreases in intrinsic growth rate were observed in C. lactis, C. redickorzevi,
and D. farinae. The maltose additive in the diet accelerated the growth rates of A. siro,
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Figure 2. The coloration of the mesodeum of specimens that ingested starch azure as a diet. The released
remazol brilliant blue (RBB) from starch hydrolysis is shown as the blue color and the increasing
concentration of the color displays increasing enzymatic activity and is marked by empty arrows. Black
arrows show spaces without RBB after dilution or absorption to the cells of the mesodeum. A: Acarus siro;
B: Aleuroglyphus ovatus; C: Chortoglyphus arcuatus; D: Dermatophagoides farinae; E: Tyroborus lini; F: Tyrophagus
putrescentiae; G: Lepidoglpyhus destructor, food bolus in the colon of the same specimen during 3 h of
observation. RBB leaves the food bolus and its concentration decreases over time. aa, anal atrium; ca,
caecum; co, colon; fb, food bolus; ic, intercolon; pc, postcolon; v, ventriculus.
Archives of Insect Biochemistry and Physiology
Archives of Insect Biochemistry and Physiology
0.16070.023
0.20570.018
0.10070.027
0.13270.036
0.18370.024
0.08470.021
0.17970.014
0.15570.018
0.22870.011
Mean7sd
b
c
ab
a
a
a
b
b
b
T
0.18770.042
0.31170.029
0.10770.015
0.12670.041
0.18670.034
0.07470.016
0.19770.029
0.18770.017
0.29170.016
Mean7sd
Wheat
a
a
ab
a
a
b
a
a
a
T
0.17170.015
0.24470.012
0.12770.024
0.13470.029
0.18870.023
0.02370.014
0.19570.025
0.20070.013
0.27370.015
Mean7sd
Maltose
ab
b
a
a
a
c
a
a
a
T
0.12570.020
0.19470.019
0.09370.035
0.13570.031
0.13270.023
0.00670.016
0.13270.024
0.19070.029
0.21270.011
Mean7sd
Sucrose
c
c
b
a
b
d
c
a
c
T
10.34
112.64
3.09
0.18
6.71
116.21
44.60
14.84
123.78
F
3.71
3.64
3.51
3.45
3.43
3.68
3.69
3.62
3.77
d.f.
ANOVA
o0.0001
o0.0001
0.035
0.908
0.001
o0.0001
o0.0001
o0.0001
o0.0001
P
The experimental diets were the control diet with the addition (5%) of wheat starch, maltose, or sucrose. The data are means and standard deviations. The letters indicate the
Turkey’s HSD groups at the P 5 0.05 significance level.
A. ovatus
A. siro
C. arcuatus
C. lactis
C. redickorzevi
D. farinae
L. destructor
T. lini
T. putrescentiae
Species
Control
Diet
Table 2. Comparison of the Growth Rates (r) of Species Reared for 21 Days on Control and Experimental Diets
Digestion of Starch Substrates and Sucrose by Mites
149
150
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L. destructor, T. lini, and T. putrescentiae in comparison to control (Table 2). Sucrose as an
additive only accelerated the growth rate of T. lini, but showed a suppressive effect on A.
ovatus, D. farinae, C. redickorzevi, and T. putrescentiae.
Inhibition of Starch Digestion by Acarbose
The kinetic study of the inhibitory effect of acarbose on wheat starch and amylopectin
digestion showed similar inhibition curves (Fig. 3). The IC50 values showed a very
similar inhibition of amylolytic activity on amylopectin and wheat starch (Table 3). The
trends of maltase inhibition curves were remarkably different from the amylolytic
inhibition and were less steep in all cases (Fig. 3). Simultaneously, the IC50 values of
maltase inhibition were higher than the amylolytic inhibition in all cases (Table 3). The
sensitivity of a-amylase and a-glucosidase to acarbose varied among the species. The
sensitivity of the species to acarbose increased from L. destructor, D. farinae, A. ovatus,
and T. lini to the rest of the species (Table 3). The ranks of the species according to IC50
differed with maltose to amylopectin and wheat starch.
Figure 3. The effect of the a-amylase and a-glucosidase inhibitor acarbose on WME. The graphs represent
competitive inhibition of hydrolysis of wheat starch, amylopectin, and maltose to glucose. The measurement
was performed with six concentrations of acarbose (nmol/mL). The pH used in the inhibition assays was 5.0
and the buffer concentration in the reaction was 0.1 M. The enzymatic activity is expressed as the percentage
of activity remaining (vi/v0 102).
maltose;
amylopectin;
wheat starch.
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151
Table 3. The Effect of the a-Amylase and a-Glucosidase Inhibitor Acarbose on Starch Hydrolyses
In Vitro
IC50
Species
A. ovatus
A. siro
C. arcuatus
C. lactis
C. redickorzevi
D. farinae
L. destructor
T. lini
T. putrescentiae
Ratio of IC50
Maltose
Amylopectin
Wheat st.
WS/A
M/A
M/WS
23.46
4.23
3.86
16.45
7.89
46.82
88.99
14.26
4.97
6.23
3.12
1.83
0.75
2.49
6.62
25.70
6.04
1.69
7.90
2.46
1.80
0.73
2.90
4.98
17.48
8.29
4.48
1.27
0.79
0.98
0.97
1.16
0.75
0.68
1.37
2.65
3.77
1.36
2.11
21.93
3.17
7.07
3.46
2.36
2.94
2.97
1.72
2.14
22.53
2.72
9.40
5.09
1.72
1.11
The IC50 values in nmol/mL describe the inhibitor concentration that caused 50% inhibition of enzymatic activity on
wheat starch, amylopectin, and maltose in WME.
Figure 4. The effect of the a-amylase and a-glucosidase inhibitor acarbose on the growth rate of the mite
species tested. Y-axis parameters are growth rates (r) and X-axis parameters are concentrations of the
inhibitor in the diet (mg/g). The parameters of linear regression models are in Table 4.
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Table 4. The Effect of the a-Amylase/a-Glucosidase Inhibitor Acarbose on the Growth Rate of the
Tested Mite Species
Linear regression model
Parameters
R2
F
df
P
a
b
Fit rc50 (mg/g)
A. ovatus
0.89
381.2
1.48
o0.0001
A. siro
0.60
191.6
1.126
o0.0001
C. arcuatus
0.32
35.0
1.73
o0.0001
C. lactis
0.24
13.9
1.45
0.001
C. redickorzevi
0.45
43.8
1.53
o0.0001
D. farinae
0.50
55.9
1.55
o0.0001
L. destructor
0.84
199.3
1.38
o0.0001
T. lini
0.71
157.2
1.64
o0.0001
T. putrescentiae
0.82
347.7
1.78
o0.0001
0.020
(0.022/0.017)
0.034
(0.039/0.29)
0.008
(0.010/0.005)
0.005
(0.007/0.002)
0.021
(0.028/0.015)
0.010
(0.013/0.007)
0.008
(0.009/0.007)
0.031
(0.036/0.026)
0.073
(0.081/0.065)
0.143
(0.138/0.148)
0.139
(0.132/0.146)
0.081
(0.075/0.086)
0.033
(0.026/0.039)
0.101
(0.084/0.118)
0.041
(0.035/0.047)
0.176
(0.173/0.179)
0.129
(0.118/0.140)
0.119
(0.111/0.126)
3.66
(3.37/3.98)
2.05
(1.71/2.39)
5.08
(3.47/9.13)
6.6
(3.71/19.5)
2.35
(1.65/3.17)
2.08
(1.51/2.78)
11.44
(10.86/15.15)
2.06
(1.71/2.46)
0.81
(0.71/0.93)
Species
The effect was described using a linear regression model (y 5 ax1b), where the parameters a and b are presented
together with the 95% confidence limit in parentheses. The rc50, the concentration of acarbose that suppresses the
growth rate to 50% compared to the control, was estimated and the 95% confidence limits for the estimation are
presented in parentheses.
The IC50 for amylopectin and wheat starch substrates maximally differed by
740%. The exception was T. putrescentiae with an IC50 that was 2.7-fold higher for
wheat starch than for amylopectin (Table 3).
The IC50 values for maltose/amylopectin or wheat starch were different among the
species. A. siro, A. ovatus, T. putrescentiae, T. lini, C. redickorzevi, and C. arcuatus had up
to 3.5-fold higher maltase IC50 values than the amylolytic IC50. It was followed by
L. destructor, which had 3.5- and 5-fold higher maltase IC50 values than the amylopectin
and wheat starch IC50 values, respectively. D. farinae showed 7- and 9.4-fold higher
IC50 values for maltose than for amylopectin and wheat starch, respectively. C. lactis
differed from all of the other species; it had very low IC50 values for starches and an
extremely high IC50 for maltase.
The bioassay confirmed the suppressive effect of the a-amylase inhibitor acarbose
on the intrinsic growth rate of all of the tested species (Fig. 4). The analysis of
covariance (ANCOVA) was significant (R2 5 0.772; F(17, 580) 5 115.4; Po0.0001) and
showed that the acarbose concentration (F 5 80.6; Po0.0001), the species (F 5 83.5;
Po0.0001), and their interaction (F 5 48.2; Po0.0001) significantly influenced the
growth rate. The effect of acarbose on the growth rate is described in Figure 4. The
rank of species sensitivity started with T. putrescentiae, followed by A. siro, T. lini,
D. farinae, C. redickorzevi, C. arcuatus, C. lactis, and L. destuctor, as clearly illustrated by
the rc50 values (Table 4).
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153
Table 5. Correlations Between the Hydrolytic Activity of the WME and SGME With Wheat Starch,
Amylopectin, and Maltose, and the Suppressive Effect of the a-Amylase/a-Glucosidase Inhibitor
Acarbose Expressed as IC50 and rc50
Starch type
Wheat starch
Variables
SGME
SGME
WME
IC50
RC50
0.106
0.776
0.653
Amylopectin
WME
IC50
RC50
0.325
0.881
0.595
0.808
0.192
0.596
0.091
0.621
0.090
SGME
0.101
0.933
0.707
Maltose
WME
IC50
RC50
0.317
0.966
0.473
0.841
0.231
0.748
0.223
0.053
0.560
SGME
0.110
0.812
0.697
WME
IC50
RC50
0.332
0.901
0.248
0.835
0.398
0.731
0.061
0.158
0.534
The IC50 values in nmol/mL describe the concentration of inhibitor at which 50% inhibition of enzymatic activity is
observed on wheat starch, amylopectin, and maltose in WME. The rc50 is the concentration of acarbose that
suppresses the growth rate of the tested mites to 50% compared to the control. The values above the shaded boxes
are the Pearson’s correlation coefficient (P) and those below the line are the determination coefficients (R2). Values
in bold are significantly different from each other at a significance level of P 5 0.05.
The inhibitory activity expressed as IC50 positively correlated with the amount of
glucose obtained from hydrolysis of wheat starch, amylopectin, and maltose by SGME
(Table 5). There were not any significant correlations between IC50 and substrate
hydrolysis by WME. In addition, both the hydrolytic activity and IC50 positively
correlated with rc50, which means that the species with higher starch hydrolytic
activities in the SGME were more tolerant to acarbose, reflected by the higher IC50
and rc50. Among the species, L. destructor showed the highest tolerance to acarbose
(Tables 3, 4; Figs. 3, 4).
DISCUSSION
The amylolytic activity was observed in all mite species tested. We found that
a-amylases and a-glucosidases recovered from mites have optimal enzymatic activity
corresponding to the acidobasic conditions in the anterior mesodeum. Also, similar
portions of liberated glucose from all the substrates by hydrolysis of WME and SGME
supported the premise that the described enzymes are produced in the anterior
mesodeum, not in the tissues. The high specific hydrolytic activities of a-amylases and
especially maltase demonstrated the importance of their synergetic activity on
liberating the glucose from the starch. The diverse inhibitory effect of acarbose on
different substrates and among species showed differences in starch utilization among
the species. The high specific activity of the tested enzymes in SGME of all the species
indicated their high allergenic potential. The species commonly occurring in cereals
were strongly inhibited by acarbose inhibiting starch digestion.
The classification of species based on the enzymatic activities formed four clusters,
where the species were grouped according to their position in relation to the first two
axes. In cluster (1), A. siro, A. ovatus, and T. lini were associated with the type of starch
substrate, with higher enzymatic activities in WME. In cluster (2), L. destructor was
associated with the same substrates but had higher activities in feces. In both clusters,
the species were associated with starch as the key nutrient. The feeding experiments
showed accelerated intrinsic growth rates on the diet containing wheat starch in
the previously named species with high hydrolytic activity on starches. In cluster (3),
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D. farinae, C. arcuatus, and C. redickorzevi were associated rather with sucrose instead of
the starch-type substrates. The species clusters were not associated with starch
nutrients in stored products. It does not exclude starches as the sources of nutrients,
but these species seem to have adapted differently from the previous ones. This is
clearly illustrated by the suppressive effect of the a-amylase/a-glucosidase inhibitor
acarbose on the population growth of the species. Acarbose reversibly inhibits the
degradation of starch-type substrates and maltose (Asano, 2003).
The biotest did not confirm that sucrose is a suitable addition to diets for
population growth but, surprisingly, did confirm that it was partly suppressive. This
result may allow for some conclusions. For example, the sucrose additive in the diets is
very available for microbial growth and high microbial growth could be negative and
suppress mite growth.
The last cluster was formed by T. putrescentiae and C. lactis with low or intermediate
enzymatic activity on both the starch-type substrates and sucrose (Fig. 3). The diet
enriched with starch accelerated the growth of T. putrescentiae and acarbose strongly
suppressed the population growth. These mite species occur in microorganism-rich
habitats, i.e., house dust, decomposing plant/animal tissues, and soils (Hughes, 1976).
Especially symbiosis of T. putrescentiae with microscopic fungi is well described (Hubert
et al., 2004). We suggest that the fungi-feeding mites having amylolytic activity are able
to utilize glycogen as a stored polysaccharide of microscopic fungi rather than very
rigid structural polysaccharide chitin (see Siepel and de Ruiter-Dijkman, 1993). C. lactis
is called the dried-fruit mite and is often found in dried fruit (e.g., plums, apricots) and
dried milk and generally is not frequently found in starch-rich plant substrates
(Hughes, 1976). In this study, we found extremely high activity for maltase in WME in
comparison to amylolytic activities and an extremely high ratio between inhibition of
maltose/starch-type substrates by the a-amylase/a-glucosidase inhibitor acarbose.
It is well documented that a-amylases from the rice weevil Sitophilus oryzae hydrolyzes
various types of starches differently (Baker and Woo, 1992). Similar results were
observed in this study on the mites. The main differences were found between
hydrolyzed (wheat starch and amylopectin) and un-hydrolyzed starches (potato and corn
starches) obtained from Sigma-Aldrich. In this study, the starches were used without
boiling to mimic the natural situation in the gut. However, it was still far from the starch
granulae present in the endosperm of grain (Baker and Woo, 1992; Burrell, 2003). In
spite of that, the rank of species according to hydrolytic activities lacked remarkable
differences among the compared starches. When the amounts of liberated glucose from
the starches were compared to wheat starch, all species showed similar values for
particular starches in WME and excrement. This indicates that the main function of the
hydrolytic enzymes that degrade starch (a-amylases and a-glucosidases) is digestion and
the enzymes are not present in high concentrations in the tissues, including the fat body.
The observed broad optimum of starch and sucrose hydrolysis with maximal
activity at about pH 5 corresponded to previously reported pH optima for A. siro and
A. ovatus (Hughes, 1950; Akimov and Barabanova, 1976). The observed pH optima of
starch hydrolysis corresponded to the pH optima of a-amylase and a-glucosidases
reported in studied acaridid mites (Matsumoto, 1965; Akimov and Barabanova, 1976,
1978; Stewart et al., 1991; Lake et al., 1991b). The correlation between the pH optima
of digestive enzymes with the physiological conditions in the gut is one of the critical
reasons to study the digestive function of enzymes. The optimal pH for starch and
sucrose digestion correlates with the known physiological conditions of the ventriculus
and caeca (Erban and Hubert, 2009), where starch digestion is localized, as indicated
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155
by the release of the remazol brilliant blue (RBB) from the labeled starch azure. The
RBB was released in the region of the caeca and ventriculus.
The a-amylase inhibitors showed various suppressive activities on the synanthropic mites. For example, a-amylase cereal inhibitors showed high suppression of
D. pteronyssinus a-amylase, while a-amylases from D. farinae, L. destructor, and
T. purescentiae were resistant (Sanchez-Monge et al., 1996). Acarus siro a-amylases were
not inhibited by the a-AI inhibitor from Phaseolus vulgaris in vitro (Kluh et al., 2005).
Unlike the inhibitors of plant origin, acarbose is of microbial origin (Actinoplanes sp.,
see Asano, 2003) and its presence in the habitats of synanthropic mites has not
been detected in natural conditions. Previously, the strong inhibitory effect on A. siro
a-amylase was observed and the addition of acarbose to the diet suppressed population
growth of mites (Hubert et al., 2005).
In this study, the synergic inhibition of mite a-amylase and a-glucosidases by
acarbose blocked the whole starch hydrolysis, which was demonstrated by blocking
hydrolysis of wheat starch, amylopectin, and maltose. The IC50 values described here
reflect the activity of enzymes. The influence of isoenzymes on the IC50 is not expected
because the species are phylogenetically very close. For the a-amylase of Blomia tropicalis
(Acari: Acaridae), the amino acid sequences showed 68% homology with a group of four
allergens from D. pteronyssinus (Acari: Pyroglyphidae) (Cheong et al., 2009).
The species that showed high starch digestion activities (A. siro, A. ovatus, and
T. lini) had very low IC50 values. In addition, these species together with T. putrescentiae
were very sensitive to the addition of acarbose to the diets. The opposite situation was
apparent in L. destructor, which showed extremely high tolerance to inhibition by
acarbose. In D. farinae, the effect of acarbose in WME was low compared to the relative
activities of digestion of starch substrates and was extremely low against maltase. When
the starch hydrolytic activities were compared with acarbose inhibitory activities
expressed as IC50 and rc50 values, we observed correlations among hydrolytic activity
of SGME, IC50, and rc50. The species with higher starch hydrolytic activities in SGME
were more tolerant to acarbose as shown by the higher IC50 and rc50 values. No
significant correlations among WME, IC50, and rc50 values were apparent. This
indicates that high production of a-amylase and/or the absence of recycling
mechanisms (Terra et al., 1996) leads to a high concentration of a-amylase in the
feces. This seems to be a successful strategy to overcome the suppressive effect of
acarbose as a model a-amylase inhibitor.
Synanthropic mites contaminate human houses, especially house dust, animal
feed, and products for human consumption. The mites are potential pests when they
directly feed on food and contaminate the environment. The contamination of human
habitats by synanthropic mites lies in allergens (Musken et al., 2003) and the
transmission of medicinally important microorganisms (Hubert et al., 2004). Mite
allergens can cause anaphylaxis and anaphylactoid reactions after the ingestion of
mite-infested food (Sanchez-Borges et al., 2005). Anaphylaxis and asthma can be lifethreatening and every year deadly cases occur (Edston and van Hage-Hamsten, 2003).
Mite a-amylases are present among the biochemically characterized allergens (group
4). IgE epitopes for Blo t4, Der p4, and Eur m4 are documented in Blomia tropicalis,
D. pteronyssinus, and Euroglyphus maynei, respectively (Lake et al., 1991a,b; Cheong
et al., 2009). Feces are known to be an important source of mite allergens (Tovey et al.,
1981). The high enzymatic activity of both a-amylases and a-glucosidases in the feces
observed in this study indicates that these enzymes are allergens of importance in a
wider spectrum of mite species.
Archives of Insect Biochemistry and Physiology
156
Archives of Insect Biochemistry and Physiology, July 2009
The digestive enzymes in mites are frequent allergens (Stewart and Robinson,
2003). Their importance on an interspecies level is affected quantitatively by an
adaptation to the ingested food source (Bowman, 1984). For example, Dermatophagoides spp. mites are adapted to protein and/or microbe diets rather than to starches,
which was shown by low amylolytic and maltase activities in both WME and feces of
D. farinae in this study. The high activity of a-amylases and a-glucosidases in storedproduct mites observed in this study also increased their allergen importance, and the
starch hydrolytic enzymes of these species (A. siro, A. ovatus, and L. destructor) should be
included in IgE screening in human populations.
The control of mites is usually accomplished through the use of organophosphate
and pyrethroid pesticides (Collins, 2006). New regulations and policies have led to a
re-evaluation and re-registration of all groups of pesticides and their active ingredients
and some products may be eliminated from the market. This has started new research
and a search for novel control strategies of house-dust and stored-product mites.
Based on the suppressive effect on A. siro, the inhibitor acarbose has been suggested as
a novel tool for the suppression of stored-product mites (Hubert et al., 2005). In this
study, we confirmed the previous results on eight more species. The high suppressive
effect of acarbose supported the possibility of using an acarbose-based control strategy
for synanthropic mites. The acarbose is a non-proteinaceous inhibitor commercially
used as an antidiabetic agent (Asano, 2003). For the construction of transgenic plants,
proteinaceous inhibitors coded by a single gene seem to be a more feasible method
than the non-proteinaceous inhibitors studied here (Franco et al., 2002). However,
recent advances in genomics and gene expression technology have made incorporation of non-proteinaceous inhibitors feasible in the foreseeable future (Kinney, 2006).
It is a realistic alternative to incorporation into transgenic plants. In addition, we found
that mite a-amylase/a-glucosidases are suitable targets for novel control strategies for
mites. In further studies, we recommend screening inhibitor activities of a wider
spectrum of a-amylase or nonspecific a-amylase/a-glucosidases inhibitors.
ACKNOWLEDGMENT
The authors thank Jitka Stara, Michael Mares, Vaclav Stejskal, and Jaroslav Smrz for
valuable comments and Sarka Tuckova and Pavel Horak for technical assistance.
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nutritive, strategia, amylases, target, acaridid, starch, digestion, control, base, synanthropic, mites, inhibitors, sucrose, suitable, importance, biologya, glucosidase
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