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Visualization of protein digestion in the midgut of the acarid mite Lepidoglyphus destructor.

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A r t i c l e
VISUALIZATION OF PROTEIN
DIGESTION IN THE MIDGUT OF THE
ACARID MITE Lepidoglyphus
destructor
Tomas Erban and Jan Hubert
Crop Research Institute, Drnovska 507, Praha 6 Ruzyne, Czechia;
Medical Center Prague, Praha 4, Czechia
The ingestion of chromogenic or fluorescent substrates for protease detection
enables the visualization of digestive processes in mites in vivo due to their
transparent bodies. The substrates for protease detection were offered to
Lepidoglyphus destructor, and the resulting signals were observed in specimens
under a compound microscope. The protease activity was successfully localized
using chromogenic substrates (azoalbumin, AAPpNA, SAAPFpNA,
elastin–orcein, SA3pNA, ZRRpNA, ArgpNA, and MAAPMpNA) and
fluorescent substrates (casein–fluorescein, albumin–fluorescein, AAPAMC,
BAAMC, ZRRAMC, ArgAMC, and AGPPPAMC). No activity was
detected using the keratin azure and BApNA substrates. In the mesodeum,
trypsin-like activity generated by hydrolysis of the BApNA substrate was not
observed, but the BAAMC substrate allowed the visualization of trypsin-like
activity in food boli in the posterior mesodeum. The results indicate that
cathepsins B, D, and G and cathepsin H or aminopeptidase-like activities are
present in the midgut of L. destructor. Among these activities, cathepsin D-like
activity was identified for the first time in the gut of L. destructor. All proteases
mentioned are produced in the mesodeal lumen and form the food bolus
together with ingested food, afterward passing through the gut to be defecated.
The method used enables the visualization of protease activities in the gut of
C 2011 Wiley Periodicals, Inc.
transparent animals. Keywords: acaridid mite; Lepidoglyphus destructor; digestion; protease;
allergen; visualization
Grant sponsor: Ministry of Education, Youth and Sports of the Czech Republic in frame of COST action
CM0804—Chemical Biology with Natural Products; Grant number: OC10019.
Correspondence to: Tomas Erban, Crop Research Institute, Department of Stored Product Pest Control
and Food Safety, Laboratory of Proteomics, Drnovska 507, Prague 6-Ruzyne, CZ 161 06, Czechia.
E-mail: arachnid@centrum.cz
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 78, No. 2, 74–86 (2011)
Published online in Wiley Online Library (wileyonlinelibrary.com).
& 2011 Wiley Periodicals, Inc. DOI: 10.1002/arch.20441
Protein Digestion in the Midgut of Lepidoglyphus destructor
75
INTRODUCTION
Acaridid mites are important producers of allergens in the human environment
(Thomas et al., 2010). The most prevalent sources of mite allergens are feces; the feces
are excreted into the environment during the entire life of the mites (Tovey et al.,
1981). Among the spectrum of allergens produced by mites, cysteine and serine
proteases are considered the most significant. These enzymes likely have digestive
functions and therefore are secreted into the gut lumen and are passed through the
gut and become feces. This sequence has been well documented for cysteine proteases
(Stewart et al., 1991, 1992). Generally, proteases, due to their enzymatic activity, are
considered important allergens because they have the ability to disrupt cellular tight
junctions, leading to the penetration of allergens through the lung epithelium (Wan
et al., 2001) and leading to the polarization of the immune response due to cleaving
the receptors of lymphocytes (Hammad et al., 2001). The localization of proteolytic
activities in the guts of mites is important and contributes to the understanding of the
digestive physiology of mites. The identification of digestive proteases in the feces
indicates that the proteases are of significant allergenic importance. Acaridid mites are
equipped with digestive proteases that act as serine proteases, e.g. trypsin (EC
3.4.21.4) and chymotrypsin (EC 3.4.21.1), and cysteine (EC 3.4.22) proteases. The
activity of proteases in seven species of acaridid mites has recently been confirmed
(Erban and Hubert, 2010b). These proteolytic activities were determined in whole
mite extracts, and therefore it was not possible to determine which proteases move into
the gut to perform digestion. The localization of the proteolytic activities of proteins in
the gut confirms their digestive activity.
The fact that the bodies of acaridid mites are thin and transparent makes them
suitable for the application of colorimetry and for the direct determination of the gut
pH using a compound microscope (Erban and Hubert, 2010b). Previously, we
visualized the digestion of bacteria (Erban and Hubert, 2008) and starch (Erban and
Hubert, 2008) in mites. In this study, we investigated the localization of proteolytic
activities in the gut of Lepidoglyphus destructor. The results offer information on the
ability of acaridid mites to digest protease substrates in vivo and indicate which
proteases are digestive.
MATERIAL AND METHODS
Experimental Mites
L. destructor (Schrank, 1871) originated from laboratory stock cultures kept in the Crop
Research Institute, Prague, Czechia. The mites were mass-reared and collected as
previously described (Erban and Hubert, 2008).
Experimental Approach
In our approach, we presumed that the substrate is digested by mites and that changes
in the color or increases in the fluorescence in the gut are a result of the activity of the
digestive enzymes. If available, two analogous types of substrates are used, both
chromogenic and fluorescent, to better describe the hydrolysis that takes place in parts
of the digestive tract of mites after the release of pNA (p-nitroaniline), AMC (7-amido4-methylcoumarin hydrochloride), fluorescein or azo dye.
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Application of Proteolytic Substrates
To localize general protease activity in the gut of L. destructor, fluorescein conjugates of
casein (C2990; Invitrogen, Eugene, Oregon) and albumin (A9771; Sigma-Aldrich, Saint
Louis, MO) were used. In addition, a chromogenic general protease substrate, azoalbumin
(A2382; Sigma-Aldrich), was used. The following pNA-bound specific substrates were
used: BApNA (Cat. No. B4875; Sigma-Aldrich), SAAPFpNA (Cat. No. S7388; SigmaAldrich), SA3pNA (Cat. No. S4760; Sigma-Aldrich), AAPpNA (Cat. No. A9148; SigmaAldrich), MAAPMpNA (Cat. No. M7771; Sigma-Aldrich), ZRRpNA (Cat. No. P-138;
Biomol, Exeter, UK), and ArgpNA (Cat. No. P-136; Biomol). Azo-dye-impregnated
collagen (Cat. No. A4341; Sigma-Aldrich) and keratin azure (Cat. No. K8500; SigmaAldrich) were used to screen keratinase and collagenase activity, respectively. In addition to
the pNA-bound substrates, we used fluorescent substrates that were bound to AMC. The
tested AMC substrates were all purchased from Sigma-Aldrich: AAPAMC (A3401),
BAAMC (B7260), ZRRAMC (C5429), ArgAMC (A2027), and AGPPPAMC (C6983).
All the substrates and their targets listed in the manufacturer’s instructions are
presented in Table 1. The chromogenic substrates and the fluorescein conjugates (0.1 mg)
were transferred to Eppendorf tubes, and at least 50 specimens with good fitness and free
from adhering food particles and feces were added. In the case of fluorescent substrates, a
0.01% (w/w) diet enriched for the appropriate substrate was prepared by dissolving the
substrate in an appropriate solvent according to the manufacturer’s instructions, followed
by lyophilization. The diets were stored in a freezer at 401C until use.
A piece of moistened filter paper was added to the cap of each tube and fixed with
a cover to prevent desiccation. The mites were removed to a glass Petri dish in time
intervals from 24 to 72 h in the case of chromogenic substrates and 30 min to 4 h in the
case of fluorescent substrates. The mites with presence of the ingested indicator in the
gut were collected using a hair pencil under a Stemi 2000-C dissection microscope
(Carl Zeiss, Jena, Germany). After being rinsed in a drop of physiological saline (0.9%
NaCl), the specimen was prepared for light microscopy in physiological saline. The
Table 1. Overview of Substrates Used for the Detection of Specific and Nonspecific Protease
Activity, According to the Manufacturers’ Instructions
Substrate
Casein–fluorescein conjugate
Albumin–fluorescein conjugate
Azoalbumin
AAPAMC
AAPpNA
SAAPFpNA
BAAMC
BapNA
Keratin azure
Elastein orcein
SA3pNA
ZRRAMC
ZRRpNA
ArgAMC
ArgpNA
AGPPPAMC
MAAPMpNA
Code
Target protease
(C2990; Invitrogen)
(A9771; Sigma-Aldrich)
(A2382; Sigma-Aldrich)
(A3401; Sigma-Aldrich)
(A9148; Sigma-Aldrich)
(S7388; Sigma-Aldrich)
(B7260; Sigma-Aldrich)
(B4875; Sigma-Aldrich)
(K8500; Sigma-Aldrich)
(E1500; Sigma-Aldrich)
(S4760; Sigma-Aldrich)
(C5429; Sigma-Aldrich)
(P-138; Biomol)
(A2027; Sigma-Aldrich)
(P-136; Biomol)
(C6983; Sigma-Aldrich)
(M7771; Sigma-Aldrich)
General
General
General
Chymotrypsin, tripeptidyl peptidase I
Chymotrypsin, tripeptidyl peptidase I
Chymotrypsin, human leukocyte cathepsin G
Peptidyl prolyl isomerase
Trypsin, Papain
Keratinase
Elastase
Elastase
Cathepsin B, trypsin
Cathepsin B, trypsin
Cathepsin H, aminopeptidases
Cathepsin H, aminopeptidases
Cathepsin D
Cathepsin G
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Protein Digestion in the Midgut of Lepidoglyphus destructor
77
color changes of the gut were observed under an Axioskop compound microscope
using Axiovision software (Carl Zeiss), and a Powershot A620 digital camera (Canon
Inc., China) was used to visualize the fluorescent substrates. Filter sets No. 02 and
No. 09 (Carl Zeiss) were used for AMC and fluorescein, respectively. The minimal
design included 10 positive observations per species and per substrate. The
methodological approach is illustrated in Figure 1. The substrate offered as food
Figure 1. Fluorescence signal in the gut of Lepidoglyphus destructor after ingestion of a fluorescent substrate
with AMC in vivo. (A) Transmission microscope view of the mite; (B) view of the same specimens using a set of
fluorescent No. 2 filters and an analyzer slide fixed for transmitted light; (C) the same without the analyzer
slide fixed for transmitted light; (D) view of food particles containing fluorescent substrate AMC, using an
analyzer slide fixed for transmitted light; and (E) the same without the analyzer slide fixed for transmitted
light. c, colon; ca, caeca; pc, postcolon; v, ventriculus; AMC, 7-amido-4-methylcoumarin hydrochloride.
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showed a fluorescence signal (Fig. 1D and E). When the slide fixed for transmitted
light was used (Fig. 1E), the intensity of the fluorescence signal was higher than
without the slide filter (Fig. 1D). A similar increase in the signal was apparent when the
mites were observed (Fig. 1B and C).
Histological Observation of the Gut
The mites were fixed in modified Bouin-Dubosque-Brazil fluid according to Smrz
(1989), embedded in Paraplast, sectioned (thickness 5–7 mm) on an HM 200
microtome (Carl Zeiss) and stained with Masson’s triple stain. The sizes of the
structures were measured for at least five specimens, and the mean is presented. The
nomenclature of the gut was adopted from Sobotnik et al. (2008).
RESULTS
Morphological Description of the Gut
Generally, the gut morphology of L. destructor is similar to that of Acarus siro (Sobotnik
et al., 2008). The foregut is formed from the pharynx and the esophagus. Visualization
with the substrates was apparent in the esophagus (Fig. 2A) but not in the pharynx.
The esophagus is 80-mm length and 15-mm width. The mesodeum consists
of a ventriculus 95-mm long and 110-mm wide. Two paired caeca are attached laterally
(Fig. 2B). The caeca are 100-mm long and 70-mm wide. The ingested food forms food
boli in the ventriculus. The size of the food bolus varies from 80 to 100 mm. The food
bolus is passed to the colon and then to the rectum. The intercolon was not
distinguished. The colon is 90-mm wide and 90-mm long. The diameter of the food
bolus is 50 mm in the colon. The postcolon is 80-mm wide and 75-mm long. The
diameter of the food bolus in the postcolon is 40 mm. Before defecation, the food bolus
is passed through the anal atrium (hindgut), in which it was not possible to visualize the
ingested substrates (Fig. 2C).
Digestion of Nonspecific Proteolytic Substrates
The substrates hydrolyzed in separate parts of the mesodeum of L. destructor are
summarized in Table 2. The albumin fluorescein conjugate was formed after ingestion
of food boli. The fluorescence signal was apparent in the whole mesodeum (Fig. 2D).
The fluorescent marker was present in the ventriculus and in the caeca and entered
into the cells of the anterior ventriculus and the anterior caeca. The marker was visible
inside the granulae of these cells (Fig. 2D and E). The fluorescent marker was also
present outside of the food boli in the colon and the postcolon (Fig. 3E). Similar
features showed chromogenic azoalbumin (Fig. 2F). The ingestion of azocasein–
fluorescein conjugate was similar to the situation observed with the albumin. The high
concentration of the fluorescein in the postcolon (Fig. 3G) was remarkable.
Digestion of Specific Proteolytic Substrates
Chymotrypsin-like proteases were identified using fluorescent (A3401) and chromogenic substrates (A9148 and S7388). The ingestion of the fluorescent substrate showed
the presence of the marker in the ventriculus and in the caeca and showed the
penetration of the fluorescent marker into the cells of the anterior ventriculus and
Archives of Insect Biochemistry and Physiology
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Figure 2. (A–C) Histological features of Lepidoglyphus destructor gut. (A) Sagittal section through the gut;
the arrows indicate the rest of the cell inside the lumen of the ventricles; (B) detailed view of the ventriculus
and the caeca; the arrows indicate the proliferated part of caecal cells; (C) detailed view of the postcolon
and the anal atrium; (D–G) visualization of nonspecific protease activities using fluorescent and
chromogenic substrates ingested by specimens of L. destructor; (D) total view of specimens that ingested
fluorescent azoalbumin; the empty arrows point to the fluorescence signal, and the black arrows indicate the
mesenteric cells containing fluorescein inside the granulae; (E) detailed view of fluorescent azoalbumin; the
arrows indicate the mesenteric cells with stained granulae; (F) total view of specimens fed A2382 azoalbumin;
the empty arrow indicates the yellow color liberated from the substrate, and the black arrow indicates
granulized mesenteric cells; (G) total view of the specimen fed fluorescent azocasein. at, anal atrium;
e, esophagus; c, colon; ca, caeca; fb, food bolus; pc, postcolon; rt, reproductive tract; syn, synganglion;
v, ventriculus. Scale: 100 mm.
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Table 2. Summary of Substrate Hydrolysis in Separate Parts of the Lepidoglyphus destructor
Mesodeum
AM
Substrate
MM
PM
v1ca
FB
Colon
FB
Postcolon
FB
1
1
1
1
1
1
Casein–fluorescein
conjugate
Albumin–fluorescein
conjugate
Azoalbumin
1
1
1
1
1
1
1
1
1
1
1
1
AAPAMC
1
1
1
1
AAPpNA
SAAPFpNA
1
1
?
?
1
1
1
1
1
1
1
1
1
BAAMC
BApNA
Keratin azure
Elastein orcein
1
1
1
1
1
1
SA3pNA
ZRRAMC
ZRRpNA
1
1
?
1
1
?
1
1
1
1
1
1
1
1
1
1
ArgAMC
ArgpNA
AGPPPAMC
MAAPMpNA
1
?
1
1
1
?
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Note
Fluorescein inside mesenteral
and cecal cells
Very high concentration
of fluorescein in postcolon
Yellow-stained granulae in
mesenteral and cecal cells
Fluorescein inside anterior
mesodeal cells, reproductive
tract and parenchymal tissue
Yellow-stained granulae inside
mesenteral and cecal cells
In restrcited area of the boli
Decrease in red color intensity
from ventriculus to colon in FB
Yellow-stained granulae inside
mesenteral and cecal cells
Yellow-stained granulae inside
mesenteral and cecal cells
Legend: The signal was present (1) or absent ( ); ? – denotes a nonexplicit result; AM, anterior mesodeum; MM,
middle mesodeum; PM, posterior mesodeum; v, ventriculus; ca, caeca.
the anterior caeca (Fig. 3A). In addition, the marker penetrated into the fat body and
the reproductive tract. The presence of the fluorescent marker outside of the food
bolus in the colon was apparent, while it was not observed outside of the food bolus in
the postcolon. The specimens feeding on both the AAPpNA and the SAAPFpNA
chromogenic substrates showed apparent yellow granulation inside the mesenteric
and caecal cells (Fig. 3B and C), and the yellow color was present in both the colon and
the postcolon outside of their food boli (Fig. 3C and D).
For detection of trypsin-like proteases, no color changes were observed in
specimens fed BApNA (B4875). The specimens fed a fluorescent substrate (B7260)
had a fluorescence signal in the colon and the postcolon only. The signal was visible
inside the food boli but was located in a restricted area of the boli (Fig. 3E).
The specimens fed SA3pNA chromogenic substrates had yellow-stained granulae
in the ventriculus and the caeca. The yellow color was apparent in the food bolus in the
postcolon (Fig. 3F). The specimens feeding on elastin–orcein had a rose color in the
whole mesodeum (Fig. 3G and H), including a rose-colored food boli. There appeared
to be a decrease in the color intensity of the food bolus from the ventriculus to the
colon (Fig. 3H). No color changes were observed in specimens feeding on the keratin
azure (K8500) substrate.
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Figure 3. (A–E) Visualization of specific proteases for the detection of chymotrypsin-like and trypsin-like
activity using fluorescent and chromogenic substrates ingested by specimens of Lepidoglyphus destructor.
(A) Total view of the specimens fed a fluorescent chymotrypsin substrate (A3401); the empty arrows point to
the fluorescent marker, the black arrows point to mesenteric cells containing substrate inside granulae, and
white arrows point to a fat body with substrate-containing granulae; (B) total view of specimens that ingested
AAPpNA (A9148); the empty arrows point to yellow color outside of the food bolus in the colon, and the
black arrows indicate yellow mesenteric cells having yellow granulae; (C) the specimens feeding on
SAAPFpNA (S7388) with apparent mesenteric cells containing yellow-stained granulae (arrows); (D) the
specimens feeding on SAAPFpNA (S7388) with an apparent yellow color outside of the food bolus in the
postcolon, indicated by the empty arrow; (E) total view of specimens feeding on fluorescent substrate B7260
indicating trypsin-like activity; the empty arrows indicate the signal in the food bolus in the colon and
postcolon; (F) total view of specimens feeding on the SA3pNA chromogenic substrate; the empty arrows
indicate the yellow color in the caeca and in the food bolus in the colon; (G, H) the specimens fed
chromogenic elastin; (G) total view; the empty arrows indicate the rose color; (H) detailed view of the
mesodeum; the empty arrows indicate the rose color, and the black arrows indicate the substrate inside the
food boli. e, esophagus; c, colon; ca, caeca; egg, egg; fb, food bolus; pc, postcolon; rt, reproductive tract; v,
ventriculus. Scale: 100 mm.
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Cathepsin B-like and/or trypsin-like activity was indicated by feeding on the
ZRRAMC and ZRRpNA substrates. The fluorescence signal on ZRRAMC was present
in the ventriculus and the caeca and inside the food boli in the colon and the postcolon.
In addition, the feces showed a high signal response (Fig. 4A). The signal was also
visible outside of the food bolus in the colon and postcolon. A similar distribution
appeared for the chromogenic substrate ZRRpNA, as after its hydrolysis, pNA
penetrated into the cells of the anterior mesodeum. The highest concentration of
yellow color was in the colon and the postcolon outside of the food boli (Fig. 4B).
Cathepsin H or aminopeptidase-like activity was indicated by feeding on ArgAMC
and ArgpNA. The fluorescence signal was present in the ventriculus and the caeca and
inside of the food boli in the colon and postcolon (Fig. 4C). The specimens feeding on
chromogenic ArgpNA developed yellow granulae inside the cells of the anterior
ventriculus and the caeca. The yellow color was apparent outside of the food boli in the
colon and postcolon (Fig. 4D).
Cathepsin D-like activity was identified by feeding on the fluorescent substrate
AGPPPAMC. The fluorescence was observed in the whole mesodeum and penetrated
through the mesodeum (Fig. 4E) to the parenchymal tissues and the reproductive
tract. The highest concentration was inside of the food boli in the colon and the
postcolon (Fig. 4F).
Cathepsin G-like activity was indicated by feeding on the chromogenic substrate
MAAPMpNA. Yellow color was apparent in the ventriculus and in the caeca and inside
of the food bolus in the colon (Fig. 4G) and the postcolon. The color entered the
anterior ventriculus and caecal cells and was visible as yellow granulae inside these cells
(Fig. 4H).
DISCUSSION
The transparent and thick body of L. destructor enabled the use of proteolytic substrates
and enabled us to localize the proteolytic activity in vivo in the mesodeum, the food
bolus, and the feces of mites. In addition, the ingestion of a fluorescent substrate
allowed us to visualize the mesodeum for morphological description and contributed
to our understanding of the digestive ability of mites. The coloration in the mesodeum
indicated the activities of chymotrypsin-like proteases and elastase but not those of
other proteins. In the mesodeum, the trypsin-like activity resulting in the hydrolysis of
the BApNA substrate has not previously been observed, but the BAAMC substrate
revealed trypsin-like activity in food boli in the posterior mesodeum. The results
indicated that the cathepsins B, D, and G and cathepsin H or aminopeptidase-like
activities are present in the midgut of L. destructor. Among these activities, cathepsin
D-like activity was found for the first time in the gut of L. destructor. The hydrolytic
activity of keratin azure has not been observed previously, either. All mentioned
proteases are secreted into the mesodeal lumen and together with the ingested food
form the food bolus, which is defecated after passing through the gut. Demonstration
of the specificity of enzymatic activities toward the substrates in vivo using enzyme
inhibitors represents a challenge for future study.
The use of chromogenic substrates for the visualization of digestive processes has
been tested previously using starch azure and Micrococcus lysodeikticus for the
localization of a-amylase and bacteriolytic activity in mites, respectively (Erban and
Hubert, 2008; Erban et al., 2009). Recently, the digestive activity of proteases in the
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Figure 4. (A–H) The visualization of cathepsin-like proteases using fluorescent and chromogenic
substrates ingested by specimens of Lepidoglyphus destructor. (A) Total view of a specimen fed the cathepsin
B (C5429) fluorescent substrate; the arrows point to sites with high concentrations of the fluorescent marker;
(B) a specimen fed the ZRRpNA (P138) chromogenic substrate; the empty arrows point to the yellow color in
the colon and postcolon, and the black arrows point to mesenteric cells containing substrate inside granulae;
(C) total view of specimens fed the cathepsin H (A2027) fluorescent substrate; the arrows point to areas with
high concentrations of fluorescent markers; (D) specimens fed the ArgpNA (P136) chromogenic substrate;
the empty arrows point to the yellow color in the colon and postcolon; the black arrows point to mesenteric
cells containing substrate inside granulae; (E, F) specimens fed the cathepsin D (C6983) fluorescent
substrate; (E) total view, with arrows pointing to areas with high concentrations of fluorescent marker; (F)
detailed view of the mesodeum, with arrows pointing to areas with high concentrations of fluorescent
markers in food boli in the colon and in the postcolon; (G, H) specimens fed MAAPMpNA, indicating
cathepsin G activity; (G) total view; (H) detailed view, with black arrows pointing to mesenteric cells that
contain substrate inside of granulae. at, anal atrium; c, colon; ca, caeca; egg, egg; e, esophagus; fb, food bolus;
fe, – feces; pc, postcolon; rt, reproductive tract; v, ventriculus. Scale: 100 mm.
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mesodeum of L. destructor was studied using whole mite extracts and both unspecific
and specific substrates (Bowman, 1981; Erban and Hubert, 2010a). For better
understanding of protein digestion in mites, it is necessary to demonstrate the
localization of the activities in the gut and/or to determine enzymatic activity in feces.
In this article, the activity was successfully localized using chromogenic substrates,
namely azoalbumin, AAPpNA, SAAPFpNA, elastin orcein, SA3pNA, ZRRpNA,
ArgpNA, and MAAPMpNA. In some cases, the yellow color was hardly visualized.
The fluorescent substrates yielded suitable results for more sensitive detection. Both
fluorescein and AMC are unaffected by pH over the range of pH 5–7 (see SigmaAldrich datasheet) that occurs in the gut of mites (Erban and Hubert, 2010b). This pH
stability enables their application in the visualization of the digestive processes of mites.
When the analyzer slide fixed for transmitted light was used, the background and
autofluorescence of mites were omitted. The switch of the slide analyzer increased the
fluorescence signal. This is a methodical approach in which we can select between
increased and decreased fluorescence, omitting autofluorescence (Fig. 1).
We found the presence of fluorescent markers in esophagus. It was in the
specimens that showed fluorescence in the ventriculus. The explanation is that the
esophagus contains regurgitated particles from ventriculus. Such situation was
observed in Acarus siro using transmission microscopy (Sobotnik et al., 2008).
Nonspecific protease substrates were digested in the whole gut, as indicated by the
presence of the liberated fluorescein. The presence of fluorescein- and yellow-stained
granulae from the chromogenic substrate inside the cells of the anterior ventriculus and
the caeca indicated that some utilization of proteins takes place. In the anterior
mesodeum and the caeca of A. siro, a high proportion of digestive cells have been
observed (Sobotnik et al., 2008). The speculation is that the digestion of proteins is
connected to these cells in the anterior mesodeum and that the yellow-stained granulae
correspond to the granules observed in digestive cells. However, the high concentration
of fluorescein in the colon and mainly in the postcolon indicates that meaningful
proteolytic activities are also present in the posterior mesodeum. This finding confirms
Akimov’s (1985) suggestion that the proteins are absorbed in the posterior mesodeum.
Here, the size of the food bolus decreased by a factor of two from the anterior to the
posterior mesodeum. It has been suggested that the reduction is correlated with water
absorption in the colon and in the postcolon. Thus, the digested proteins are absorbed
together with water through the cells of the posterior mesodeum.
Remarkably, when fluorescein-bound casein and albumin were compared, a high
concentration of fluorescein was observed in the postcolon from casein. The pH
profiling of azocasein and azolabumin provides an explanation. The extract of
L. destructor shows the highest level of hydrolysis of azocasein at pH 6, which
corresponds to the pH in the postcolon, while for azoalbumin, the highest level of
hydrolysis is at pH 5, corresponding better with the conditions in the anterior or
middle mesodeum (Erban and Hubert, 2010a,b).
The visualization of digestion using specific substrates showed different results.
Chymotrypsin-like activity was observed in the whole mesodeum using AAPAMC,
AAPpNA, and SAAPFpNA substrates. This result confirms the results of in vitro
analyses, which suggested that chymotrypsin-like enzymes are digestive (Erban and
Hubert, 2008). Hydrolysis of the BApNA and BAAMC substrates indicate trypsin-like
activity. No activity was found based on BApNA, and this observation confirms the in
vitro results, where the highest activity was at a pH outside of the conditions found in
the mesodeum (Erban and Hubert, 2008). The specimens fed BAAMC had a
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fluorescence signal in the food bolus in the colon and postcolon. Possible explanations
are (i) the enzyme hydrolyzing the substrate is produced in an inactive form and is
activated in the food boli in the colon and postcolon; and (ii) the proteolytic activity is
caused by microorganisms in the food bolus. It is well known that living microorganisms are present inside the guts of mites (Valerio et al., 2005; Sobotnik et al., 2008).
The activity of elastase was visualized by elastin-orcein and SA3pNA and was present
in all parts of the mesodeum. The observed decrease in the elastin–orcein signal between
the anterior and the posterior mesodeum indicates that this enzyme is produced in the
anterior ventriculus and that utilization takes part in the whole mesodeum.
Among the cathepsins, the liberation of a fluorescence signal from AGPPPAMC
indicates cathepsin D-like activity, which was observed in the whole mesodeum. In the
acaridid mite, Cathepsin D activity was only detected in body extracts of Acarus farris
and Tyrophagus putrescentiae, indicating that this enzyme is not a digestive protease
(Ortego et al., 2000; Sanchez-Ramos et al., 2004); nevertheless, cathepsin D-like
aspartate proteases are considered to be major digestive endopeptidases of Psoroptes
cuniculi (Nisbet and Billingsley, 2000). In this study, it was observed that hydrolytic
activity against AGPPPAMC occurs in the mesodeum, indicating possible digestive
cathepsin D-like proteases in L. destructor. Nisbet and Billingsley (2000) discuss the
possibility that cathepsin D activity is connected to lysosomes and that this enzyme may
be involved in intracellular digestion in the ventriculus. We suggest that this enzyme
may also be involved in extracellular digestion after degeneration of the ‘‘digestive
cells’’ or following exocytosis. In L. destructor, the activity was observed in the whole
ventriculus and caeca and inside the food boli in the colon and the postcolon (Fig. 3E
and F). Although these results do not exclude the suggested mechanisms for Psoroptes
cuniculi, the rest of the degenerative cells is found frequently in the mesodeum of
L. destructor, as is illustrated in Figure 1A and B.
The substrates for detection of cathepsins B, D, and cathepsin H or aminopeptidase revealed the presence of markers of hydrolysis in the whole gut without any
apparent compartmentalization. This result confirms their digestive function, as was
suggested based on the in vitro observation (Erban and Hubert, 2008).
ACKNOWLEDGMENTS
The study was supported by the project No. OC10019; COST action CM0804; Chemical
Biology with Natural Products. We thank Klara Sulcova for her technical help.
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