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Changes in integument histology and protein expression related to the moltingcycle of the black tiger shrimp, Penaeus monodon
Waraporn Promwikorn, Pornpimol Kirirat, Pranom Intasaro, BoonsirmWithyachumnarnkul
PII: S1096-4959(07)00158-3DOI: doi: 10.1016/j.cbpb.2007.04.009Reference: CBB 8860
To appear in: Comparative Biochemistry and Physiology
Received date: 20 March 2006Revised date: 4 April 2007Accepted date: 10 April 2007
Please cite this article as: Promwikorn, Waraporn, Kirirat, Pornpimol, Intasaro, Pranom,Withyachumnarnkul, Boonsirm, Changes in integument histology and protein expressionrelated to the molting cycle of the black tiger shrimp, Penaeus monodon, ComparativeBiochemistry and Physiology (2007), doi: 10.1016/j.cbpb.2007.04.009
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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CBP ms.12060 Revised – part B
Changes in integument histology and protein expression related to the
molting cycle of the black tiger shrimp, Penaeus monodon
Waraporn Promwikorn1*, Pornpimol Kirirat1, Pranom Intasaro1, and
Boonsirm Withyachumnarnkul2
1 Department of Anatomy, Faculty of Science, Prince of Songkla University, Hat Yai,
Songkhla, Thailand, 90112. 2 Department of Anatomy and Center of Excellence for Shrimp
Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok,
Thailand, 10400.
*Correspondence:
Waraporn Promwikorn, Department of Anatomy, Faculty of Science, Prince of Songkla
University, Hat Yai, Songkhla, Thailand, 90112, Tel. +66 74288135, Fax. +66 74446663,
E-mail address: [email protected]
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Abstract
We investigated changes in the histology and protein expression in the epidermis and
sub-epidermis of the black tiger shrimp (Penaeus monondon) during the molting cycle. The
epidermis consists of a cell layer located beneath the cuticle, while the sub-epidermis is
mainly composed of sub-epidermal cells and tegumental glands. During the molting cycle,
the epidermal cells increase in cell height and number, and the sub-epidermis increases in its
storage of carbohydrate, protein, mucus, and other unidentified substances at the time of the
active period of cuticular regeneration. At the early premolt (stage D0), the epidermal cells are
tidily organized, but short. Storage of carbohydrate and protein in the sub-epidermis is not
observed. During the rest of the premolt (D1- 4 stages) and the early postmolt A stage,
epidermal cell height and sub-epidermal deposition are increased, and reached a maximum
during the D4 to A stages. The period of late postmolt stages B-C3 is the time for a decrease
in epidermal cell height and sub-epidermal depositions. Lastly at intermolt stage C4, the
epidermal cells become short, and untidily organized. Sub-epidermal deposition is not
observed. Protein expression in the epidermis and sub-epidermis was observed by SDS-
PAGE. This revealed that the profile of a protein band with a molecular mass of 57 kDa
corresponded with the profile observed by histochemistry. All results point to the conclusion
that both the epidermis and sub-epidermis play major roles in cuticular regeneration. It may
also reflect the level of metabolic activity of the integument during the molting cycle. In
addition, for the first time, this work provides direct evidence of the epidermal and sub-
epidermal changes that occur during the molting cycle of the black tiger shrimp.
Keywords: histology, integument, molting cycle, Penaeus monodon, SDS-PAGE
1. Introduction
In crustaceans, post-embryonic growth and development are characterized by a
molting cycle. Each molting cycle has various stages including pre-ecdysis (premolt, stages
D0-4), ecdysis (stage E), post-ecdysis (postmolt, stages A1-2, B1-2 and C1-3), and intermolt
(C 4) stage (Drach, 1939; Skinner, 1962). The progression within each molting cycle is
regulated by the balance of the molt-stimulating ecdysteroid, and the molt-inhibiting
hormones. The main purpose of the molting process is to shed the cuticle to allow for an
increase in body mass. A number of organs and tissues including the epidermis are
responsible for this crucial task.
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The epidermis (sometimes referred to as ‘hypodermis’) is located underneath the
cuticle, and is a target for ecdysteroids (Traub et al., 1987; Ueno et al., 1992). The epidermis
is directly involved in the resorption and secretion of organic and inorganic materials from and
to the cuticle (Roer and Dillaman, 1984; Machado et al., 1990; Ziegler, 1996; Hagedorn and
Ziegler, 2002; Pratoomchat et al., 2002). During these tasks, the cellular activities of the
epidermis change dramatically depending on the stage of the molting cycle. The size of the
epidermal cells increases during the premolt, and diminish during postmolt stages, while the
sub-epidermal tissue stores lipoprotein for the developing cuticle of lobsters and crabs (Travis,
1955; Skinner, 1962; Green and Neff, 1972; Schultz and Kennedy, 1977). Changes in the
epidermal DNA, RNA, and protein content of lobsters and crayfish during the premolt period
have also been reported (Skinner, 1966; McWhinnie and Mohrherr, 1970; Humphreys and
Stevenson, 1973; Wittig and Stevenson, 1975; Dall and Barclay, 1979). The overall findings,
from both the histological and molecular levels of the epidermis, have led to a better
understanding of the molting mechanisms of crustaceans. However, this knowledge has been
obtained using various crustacean species, and the finer details of the mechanism of any one
organism, that are essential for advanced research, are still unknown.
We have been gathering information on the regulatory mechanisms of the molting
cycle in the black tiger shrimp (Penaeus monondon), an important agricultural export product
of Thailand and some other countries around the world. We recently reported on the criteria
used for determining the molting stages of this particular species (Promwikorn et al., 2004)
and on the histological characterization of connective tissue fibers, carbohydrate, protein,
lipid, and calcium salt deposited in the cuticle throughout the molting cycle (Promwikorn et
al., 2005). The aim of this study is to investigate the changes in histology and protein
expression of the integument that occur during the molting cycle in order to achieve a better
understanding of the molting mechanisms of this species. For the first time, this work
provides direct evidence on the cuticular, epidermal and sub-epidermal changes that occur
during the molting cycle of Penaeus monondon. The findings of this study are likely to apply
to various other species and be of use for more advanced studies.
2. Materials and methods.
2.1. Animals
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Healthy black tiger shrimps (P. monodon) were obtained from commercial farms.
During transportation to our laboratory in PSU, the shrimps were continuously oxygenated.
The age of the collected shrimps was estimated at 90 days, and their body mass averaged 15 g.
2.2. Shrimp culture
Natural sea water, used in all experiments, was stored in a tank for at least 2 weeks
before use. Some days before the experiments, the sea water in the aquarium was
continuously aerated. The salinity was adjusted to be similar to the sea water used in the
farms (10-30 ppt. depending on the farm). The molting stage of each shrimp was determined
prior to its cultivation. The shrimps were fed three times a day with food pellets. Natural day-
light (12:12 h cycle) and atmospheric temperatures (approximately 28 oC at night and 34 oC
during the day-time) were used throughout the experiment in order to mimic natural culturing
conditions in Thai farms.
2.3. Determination of molting stages
Daily physical examinations were made to determine the molting stages. Briefly, each
shrimp was gently picked by hand. The uropods were then quickly examined with a light
microscope. The physical criteria used for determining the molting stage are illustrated in
Figure 1. After a 1–2 min examination, the shrimps were either placed back into the
aquarium, or sacrificed. For every shrimp, the molting stage was always confirmed by
histological criteria.
2.4. Histoloy
The cuticular tissues at the carapace and trunk (first abdominal somite) of at least 10
shrimps at each molting stage were dissected and immediately fixed in Davidson’s fixative to
eliminate any calcium salt in the cuticle. The fixation stage was continued for 72 h at room
temperature with a daily change of fresh fixative. The cuticular tissues were subsequently
dehydrated in increasing concentrations of ethanol that ranged from 50 to 100%, and prepared
for routine histological embedding in paraffin blocks. Sections (5 µm thick) were cut,
paraffin was removed, and sections stained with either modified Masson’s trichrome (aniline
blue, Ponceau S, hematoxylin) (Bancroft and Gamble modification, 2002a), or periodic acid
Schiff reagent (PAS) and hematoxylin (Bancroft and Gamble modification, 2002b). The
stained tissue sections were examined with a light microscope (Olympus BX 51) and
photographed with a digital camera (Olympus DP11) connected to the microscope.
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2.5. Protein expression.
Each epidermal sample was removed from the carapace of a shrimp, and frozen at -80
oC. The sample was individually homogenized on ice with 200 µL lysis buffer (10mM Tris,
pH 8.0, 40 mM Na4P2O7, 50 mM NaF, 5mM MgCl2, 100 µM Na3VO4, 10mM EGTA, 1%
Triton-X 100, 0.5% C24H39O4Na, 1 mM PMSF, 20 µg/mL leupeptin, 20 µg/mL aprotinin).
The homogenate was then frozen with liquid nitrogen, thawed (4 oC, 10 min), and centrifuged
(10,956 x g, 4 oC, 15 min) in order to precipitate the cell debris. Supernatant (150 µL) was
transferred to a new tube. The protein concentration of the supernatant was determined with
the DC protein assay (Bio-Rad) following the manufacturer’s instructions, using bovine serum
albumin as the reference protein. Before fractionation, 10 µg protein was mixed in a 1:1
dilution with a mixture of Laemmli sample buffer (Bio-Rad) and 2 β−ME (20:1 dilution), and
boiled (10 min). The samples (a set of eight, D0-B2) were then electrophoretically separated
on a 8.6 x 6.8 cm, 10% polyacrylamide gel. Electrophoresis was performed at a constant 10
mA in a running buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS) at room temperature
until the dye front almost reached the bottom of the gel. The protein bands were visualized by
staining with either Coomassie blue R-250 or silver nitrate. For silver staining, the gel was
fixed in 40% ethanol and 10% acetic acid for 30 min, followed by sensitizing with 30%
ethanol, 0.125% glutardialdehyde, 12 mM Na2O3S2, 0.8 M Na-acetate for 30 min and washing
three times for 15 min in de-ionized water. The gel was immersed in 0.25% AgNO3 for 20 min
and then rinsed twice for 2 min in de-ionized water. The development stage occurred in 235
mM Na2CO3 and 0.0074% formaldehyde (37%). Finally the reaction was terminated with 39.2
mM EDTA-Na2. The gel image was scanned with a scanner (ImageScannerTM, Amersham
Biosciences) driven by MagicScan 32 software (version 4.6). For analysis of the gel image,
ImageQuant TL software (version 2003.03, Amersham Biosciences) was used.
2.6. Statistical analysis
Quantification of the protein bands was performed by densitometry on three
independently performed experiments. The statistically significant differences among means
of each protein band of all 8 molting stages was determined using one way analysis of
variance (ANOVA). Tukey’s HSD test was used to determine significant differences between
groups.
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3. Results
3.1. Identification of the molting stages.
Identification of the molting stage is based primarily on the physical characteristics of
the cuticular tissue as shown in Figure 1. In the intermolt stage, the setal cones (SCs) of
shrimps are fully developed and arranged in a neat line at the base of the setae (S). The
epidermal tissue (E), which is located interior to the cuticle, spreads throughout the area
beneath the cuticle and between pairs of SCs (stage C, Fig. 1a). Once the epidermal tissue
begins to retract from the cuticle, the shrimp is identified as being at stage D0 of its molt. An
observed clear straight margin of the epidermal tissue (EE) at the base of the SCs determines
the ending of the D0 stage (Fig. 1b). Stage D1 is defined by the appearance of a narrow clear
zone (*) between the SCs and the epidermal tissue (Fig. 1c). The clear zone is the site for the
formation of a new cuticle. In stage D2, the width of the clear zone between the SCs and the
epidermal tissue increases, and a wave-like pattern appears at the edge of the epidermal tissue
(Fig. 1d). Small and thin fiber-like projections between the SCs and epidermal tissue are also
observed with the microscope, although they cannot be clearly seen in the micrograph. These
projections would later become new setae. Stage D3 is identified , when the clear zone
between the SCs and the epidermal tissue has clearly widened, a thin white layer (L) at the
edge of the epidermal tissue appears together with a sharp-wavy edge of the epidermal tissue
(Fig. 1e), and the fiber-like projections are easier to see with the microscope. At the last
stage, D4, of the premolt, the synthesis of new cuticle continues until the clear zone between
the SCs and epidermal tissue is extremely clear and dominant. The typical characteristics of
the epidermal tissue are now clearly marked. As a result of the white layer reflecting the light
at its edge, sharp and serrated notches are clearly observed and young setae (Sn) are clearly
seen projecting from the impressions between the sharp notches. The pigments in the
epidermal tissue are obviously indented and arranged in parallel bands (I). The SCs then start
to deform (Fig. 1f). Once stage D4 is reached, the shrimp will shed the exuvia within 24 h.
The exuvia is composed of two pieces. The major piece consists of most of the shrimp cuticle
including the ventral part of the head, the trunk, the tail and all appendages. The minor piece
is composed of carapace and rostrum. After ecdysis the shrimps immediately enter the
postmolt stage A during which the new cuticle and the setae (Sn) are very soft and delicate.
The SC is not yet seen (Fig. 1g). Only a few hours after ecdysis, the shrimps enter the
postmolt B stage during which the cuticle is hardening, and young SCs are developing.
Different sizes of SCs (SCn) are thus observed at the base of the setae (Fig. 1h). When the
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development of the SCs is completed, the shrimps enter stage C (Fig. 1a). The average timing
of the whole molting cycle is 9-12 days with the premolt stages lasting for 6-7 days, the
postmolt stages for 2-3 days, and intermolt stages for 1-2 days (Fig. 2).
3.2. Histology of the cuticle related to the molting cycle.
After the molting stages were identified by their physical characteristics, the cuticle of
the shrimps from each molting stage was examined histologically. After PAS and
hematoxylin staining, the mature cuticle of the shrimp in the intermolt stage (C4) shows four
distinctive layers based on the texture and characteristics of the tissue (Fig. 3a). The outer
(first) layer is thin and stains a deep magenta. The second layer stains dark blue with
hematoxylin, and consists of a series of alternating light and indented lamellae. The third
layer is stained pink with PAS, and characterised by fine lamella. The fourth layer has a
similar texture to the third layer, but can be distinguished in a darker staining reaction with
hematoxylin. The first layer stains red, while the rest stains blue with slightly different
shading after staining with modified Masson’s trichrome (Fig. 3b). During the early-mid
premolt period (stages D0-2), the first, second and third layers, but not the fourth layer, are
still well-recognized with PAS and hematoxylin staining (Fig. 3c). In stage D3, a fifth layer
appears next to the third-fourth layer (Fig. 4d). In stage D4, a sixth layer is observed interiorly
to the fifth layer (Fig. 3e). Immediately after ecdysis, during the A stage, only the first and
second layers, that were previous fifth and sixth layers, can be seen in the intact cuticle (Fig.
3f). Ten hours after ecdysis when the shrimps are in stage B1, a small third layer of newly
secreted cuticle is just visible (Fig. 3g). A day after ecdysis, the thickness of the third layer
increases (Fig. 3h). The shrimps are now in the B2 stage. The thickness of the third layer
further increases, and the fourth layer is synthesized as shown in Figure 3a.
3.3. Histology of the epidermis related to the molting cycle.
In general the epidermis is composed of cells organized into a single layer beneath the
cuticle. The organization and height of the epidermal cells change throughout the molting
cycle (Fig. 4). (The gap that is seen occasionally between the cuticle (C) and epidermal cells
(E), is caused by the sliding of the cuticle away from the epidermis during the histological
procedures). During the intermolt stage the epidermal cells (E) have an untidy organization,
and have an average height of 3.12 µm (Fig. 4a). During the premolt period, the cell height
gradually increases. Thus the shape of the cell changes to columnar (Fig. 4b-d). The average
cell height during stages D2-4 (mid-late premolt stages) is 8.75 µm, approximately 2.8 times
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higher than that of the intermolt stage. Their arrangement also becomes much tidier. After
shedding of the exoskeleton, the epidermal cell height gradually decreases (Fig. 4e-f). The
average cell height at stages B1-2 is 7.33 µm.
3.4. Histology of sub-epidermal tissue related to the molting cycle.
The sub-epidermis (SE) is located just inside the epidermis, and can only be detected
in some regions of the shrimp, including the carapace and the free edge of the cuticle
wrapping the trunk. PAS and hematoxylin staining shows that the sub-epidermal tissue is
mainly composed of sub-epidermal (SE) cells, tegumental glands and some other connective
tissue cells (Fig. 5). The SE cells are oval shaped and sit closely together. In some molting
stages, the cytoplasm is stained pink after PAS and hematoxylin (Fig. 5c-f). However, in
certain molting stages the cytoplasm of the SE cell is un-stained (Fig. 5a, b). Two types of
tegumental glands are distributed among the SE cells throughout the sub-epidermis. A
tegumental gland type A (Ga) is strongly stained with PAS and hematoxylin (Fig. 5b). After
staining with toludine blue the semi-thin section, shows many dense granules within the gland,
except at the nuclear (N) region (Fig. 5g). The boundaries of the cells are not clearly
delineated. A tegumental gland type B (Gb) also stains positively with PAS and hematoxylin,
but less intensely. The contents of the cytoplasm are apparently mixed with mucus throughout
the gland (Fig. 5d-e). When the semi-thin section is stained with toludine blue, its cytoplasm
stains a pink colour instead of blue (metachromasia reaction), but the area occupied by the
mucus (Mu) is left un-stained (Fig. 5h). The nucleus (N) is located at the base of the cell.
Unlike the type A gland cells, the boundaries of the type B cells are clearly observed. During
the period of the intermolt (Fig. 5a) and the early period of the premolt (stage D0, Fig. 5b), SE
cells cannot be easily identified, as its cytoplasm does not stain with PAS. Both types of
tegumental glands are present. During the period from the early-mid premolt (stage D1, Fig.
5c) to the early postmolt stage (stage A, Fig. 5e), the SE cells are easily recognized, because
the cytoplasm is positively stained with PAS, while the nucleus is enlarged and lightly stained.
The tegumental glands increase in size and number. In the late postmolt (late B stage), the
number of PAS stained SE cells dramatically decreases (Fig. 5f). The tegumental glands also
show a dramatic decrease in both size and number. When the sub-epidermis is stained by the
modified Masson’s trichrome method, the SE cell stains red with Ponceau S (Fig. 6). The
number of Ponceau S positively stained cells increases from stage D1 through to the A stages
(Fig. 6c-e), decreases in stage B (Fig. 6f), and is diminished in the intermolt and D0 stages
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(Fig. 6a, b). A summary of all the morphological features observed in each molting stage are
presented in Table 1.
3.5. Protein expression at different stages of the molting cycle.
Protein-extracts from the epidermis at different stages of the molting cycle were
subjected to electrophoresis to determine expression profiles. After staining with Coomassie
blue R-250 (data not shown) or silver nitrate (Fig. 7a) most protein bands show consistent
expression. However, a group of proteins with molecular masses of 61, 57, 44, 38, 34, 31, 13
and 12 kDa appear to be differentially expressed during the molting cycle. The intensities of
these 8 protein bands through all the molting stages were therefore measured. Of these 8
protein bands only protein band number 2 (57 kDa) showed differences that were significant
during the molting cycle (P<0.001) when three set of data were analyzed with one way
ANOVA. On further comparison among the stages with the Turkey’HSD test, the differences
of the mean intensity of band 2 between stages D2, D3, D4 or A versus B or C are statistically
significant (p<0.05). The differences of mean intensities between stages D4 or A versus C are
highly statistically significant (p<0.01) (Fig. 7b). A comparison of these stages in terms of
their percentage of total intensity was also performed. The average intensity of band 2 is at its
lowest of 1.97 % of the total, at the intermolt C stage. It increases to 8.56 % at D0 (4.3 fold
increase from stage C), 11.72 % at D1 (5.9 fold increase from stage C), 17.74 % at D2 (9.0
fold increase from stage C), 17.11% at D3 (8.7 fold increase from stage C), 18.63 % at D4
(9.5 fold increase from stage C), and 20.68 % at A (10.5 fold increase from stage C), at which
time it reaches its highest level. It then quickly decreases to 4.20 % at the B stage (4.9 folds
decrease from stage A or 2.1 folds increase from stage C) (Fig. 7b).
4. Discussion.
4.1. Determination of the molting stages.
An accurate and consistent determination of the molting stages is essential for the
study of the molting cycle. Many criteria have been used by other workers for the precise
determination of the molting stages of various crustaceans (for review, see Promwikorn et al.,
2004). In this work we have used both the physical and histological criteria of the cuticular
tissue to determine the molting stages of the black tiger shrimp. The physical criteria have
been modified from the criteria described for the brachyuran Cancer pagurus by Drach
(1939), and the histological criteria have been modified from Skinner’s (1962) descriptions for
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the land crabs Gecarcinus lateralis (Stevenson, 1985). Physical examination is a quick and
non-lethal method. It is suitable for distinguishing molting stages daily in live shrimps,
especially during the premolt stages. However, this method is not suitable for distinguishing
stages B2, C1, C2, C3 and C4, as in these stages any differences in the physical characteristics
of the cuticular tissue are difficult to observe. Histological examination of the cuticle is
therefore introduced as an additional method.
After PAS and hematoxylin staining, each of the four layers from exterior to interior,
an epicuticle (the first layer), an exocuticle (the second layer), an endocuticle (the third layer)
and a membranous layer (the fourth layer) show consistent changes and develop unique
characteristics. The histological method can differentiate the late postmolt stages, B2-C1-C2-
C3, from the intermolt stage, C4. The cuticle of the shrimp at the intermolt stage has an
attached membranous layer, while the cuticle of the shrimp at the postmolt stage has not.
However, this method is not suitable for distinguishing the early premolt D0, D1 and D2
changes from each other. In short, physical and histological methods have their-own
advantages and disadvantages. Application of both methods together does help to accurately
and consistently identify the molting stages for Penaeus monondon.
4.2. Changes in epidermal histology throughout the molting cycle.
The epidermal cell morphology actively changes throughout the molting cycle. It
changes from being short and untidily arranged in the C4 stage to a taller and tidier
arrangement during the premolt stages, simultaneously with the appearance of a newly
synthesized pre-ecdysial cuticle (epicuticle and exocuticle) underneath the old one. The
epidermal cell height gradually decreases during postmolt, concurrently with the presence of
the post-ecdysial cuticle (endocuticle and membranous layer). The height is least at the
intermolt stage, during the time when all cuticular layers are present. These events reflect that
an increase in epidermal cell height is related to the active period of cuticular regeneration
(stages D3 – C3). We suggest that the early-mid premolt period (stages D0 – D2) is the time
when the epidermal cells prepare for cuticular synthesis. The late premolt stages D3-4 are the
time for pre-ecdysial cuticle synthesis. The postmolt stages B-C3 are the time taken for
generation of the post-ecdysial layers. During the synthesis of the new cuticle, the epidermal
cells may have increased intracellular activities, such as DNA replication, RNA transcription,
protein synthesis and calcium dynamics as reported in other crustacean species (Skinner,
1966; McWhinnie and Mohrherr, 1970; Humphreys and Stevenson, 1973; Wittig and
Stevenson, 1975; Dall and Barclay, 1979; Wheatly et al., 2002; Ahearn et al., 2004; Luquet
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and Marin 2004), thus the epidermal cells increase in size. When the rate of the synthesis
declines, the size gradually decreases, and becomes shortest, when the cuticular regeneration
process is complete.
4.3. Changes in carbohydrate and protein deposition in the sub-epidermis.
It has been previously shown with other crustaceans, but not shrimp, that the sub-
epidermal tissue stores lipoprotein and carbohydrate for the developing cuticle (Travis, 1955;
Skinner, 1962; Green and Neff, 1972; Babu et al., 1985). As the cytoplasm of the SE cell
stained pink with PAS, and red with Ponceau S, this indicates that the SE cells may serve as
the stores for carbohydrate and protein. The storages apparently increase at the same time as
the active period of new cuticle regeneration (stages D3 – C3). In other crustacean species,
many types of tegumental glands can be found in the sub-epidermis (Stevenson 1985).
However, details of those glands have not been clearly described. In P. monodon we find two
obvious types (A and B) of tegumental glands after PAS staining. Both tegumental gland
types contain carbohydrate, as they are PAS positive. Type A tegumental gland also contains
numerous protein granules. Type B tegumental gland also contains mucus and substances
probably such as glycosaminoglycans or mucopolysaccharides that cause a metachromasia
reaction. In other species; polyphenol oxidase (Stevenson, 1961), tyrosinase (Stevenson and
Schneider, 1962), and mucopolysaccharides (Dall, 1965; Stevenson and Murphy, 1967;
Shyamasundari and Hanumantha, 1978) have been found in the tegumental glands. Increases
in stain-intensity and the number of glands during stages D1 to A indicate a period of high
activity for these glands. Thus, like SE cells, the tegumental glands should participate in the
production of new cuticle. These events occurr a little earlier than the existence of the new
cuticle, that starts to be seen at D3 stage. We postulate that at the D1 stage, the sub-epidermis
starts the synthesis and deposition of essential organic compounds, and later exports them to
be part of the new cuticle seen at D1. The deposition continues until the C1-2 stages, when
cuticle synthesis ceases.
4.4. Changes in protein expression in the epidermis and sub-epidermis.
SDS-PAGE has revealed that a 57 kDa protein is the only protein that is
significantly differently expressed in different stages of the molting cycle (Fig. 7). It has a
similar protein expression profile to that shown by histochemistry (Fig. 6). This strongly
indicates that the 57 kDa protein participates in the mechanism of molting, and its expression
is specially required for the progression of the premolt. Epidermal proteins and genes related
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to molting have been reported in various species. The proteins include: i) cryptocyanin protein
with masses of 88, 85 and 74 kDa found in crab Cancer magister (Terwilliger et al., 1999), ii)
N-acetyl-β-glucosaminidase with a mass of 89 kDa in the fiddler crab, Uca pugilator (Zou
and Fingerman, 1999), iii) an actin protein with a mass of 42 kDa in the Bermuda land crab G.
lateralis (Varadaraj et al., 1996), iv) unidentified proteins with masses of 26-32, 29, 35-40, 44,
46-47 kDa in the white shrimp Penaeus vannamei (Cariolou and Flytzanis, 1994), and 4
unidentified proteins with molecular masses of 15, 18, 36 and 50 kDa in the crayfish Astacus
leptodactylus-I (Bielefeld et al., 1986). The genes implicated include: i) three isoforms of an
ecdysteroid-induced gene; E75A, C and D, encoding a 90 kDa protein in shrimp (Metapenaeus
ensis) (Chan, 1998; Kim et al., 2005), ii) postmolt stage specific genes; DD4 (encoding a 57
kDa protein), DD5 (encoding a 135 kDa protein), DD9A (encoding 113 amino acids) and
DD9B (encoding 126 amino acids), iii) a chitinase gene (Pjchi-2) encoding a 59 kDa protein
in the Kuruma prawn Penaeus japonicus (Watanabe and Kono, 1997; Endo et al., 2000;
Watanabe et al., 2000; Ikeya et al., 2001), iv) a prophenoloxidase-activating factor gene
encoding a 38.8 kDa protein (Buda and Shafer, 2005), and v) cuticle genes CsAMO9.3, 13.4,
16.3, 16.5 and CsCP6.1, 14.1, 15.0, 19.0 encoding 100 - 163 amino acid proteins in the blue
crab, Callinectes sapidus (Faircloth and Shafer, 2007). After consideration of the above lists
of proteins and genes in terms of both molecular mass and expression profiling, we are not
convinced that any of them might have a similar function to the 57 kDa protein found in our
experiments. However, the two most possible candidates according to size may be, first, the
57 kDa protein encoded by the DD4 gene, but its mRNA is highly expressed in the postmolt
stage (Endo et al., 2000), and this is different from our result. Secondly, it could be the 59
kDa protein, encoded by the chitinase gene (Pjchi-2), however, its gene expression is high at
stage D3, but low at the D4 stage (Watanabe and Kono, 1997). We are not convinced that the
57 kDa protein could be a chitinase, as its gene stops being expressed at the D3 stage, which is
too early to be related to our findings. In order to establish the identity of the 57 kDa protein,
we continue to investigate the peptide mass fingerprint of the 57 kDa protein from the 1D
SDS-gel with a MALDI-TOF mass spectrometer (Reflect IV, Bruker). This procedure was
performed at the Bio-Service Unit, National Science and Technology Development Agency.
The result is shown in Figure 8. We believe that there is more than one protein candidate. At
this stage, some peptide spectrums; 861.533, 1207.94, 1477.021, 1898.239, have been selected
for sequencing by the Post-Source Decay method using Bio-Tool software (Bruker). They are
composed of amino acids KSEMG, TGLNPGKG, MSHAYAALR, and
APGPCETRFTANSR, respectively. We have tried to match these peptides to the global
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databases such as NCBI, SwissProt, and MSDS. So far, no convincing protein candidates
have been found. Further attempts to identify the protein candidates are continuing.
Meanwhile, the IDs of proteins that are expressed during the molting cycle are also being
investigated using 2D SDS-PAGE and a proteomics approach.
Acknowledgements
This work is financially supported by the Commission on Higher Education and
Thailand Research Fund through the contract number MRG4680061. We specially thank
Dr. Brian Hodgson for critical proof on the manuscript and Atchara Makatan, research
student, for technical help.
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Figure legends
Figure 1 Identification of molting stages. The criteria used for molt staging were modified
from Drach’s staging. The images of the physical characteristics indicate (a) intermolt stage,
(b-f) premolt stage and (g, h) postmolt stage. E, epidermis; EE, epidermal edge; I, indent
pattern of the epidermis; L, white layer at the edge of the epidermis; S, setae; SC, setal cone;
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SCn, newly-formed setal cones; Sn, newly-formed seta; = wavy edge of epidermis; * =
clear zone between cuticle and epidermis. All figures are with the same magnification.
Figure 2 Timing of the molting cycle. One hundred P. monodons, aged 90 days, were
cultured in aerated aquarium in natural day-light (12:12 h cycle) and at atmospheric
temperatures (approximately 28 oC at night-time and 34 oC at day-time). A molting cycle
takes a period of 9-12 days. Averaged duration of each stage was obtained from 10 -12
shrimps culturing in three continued molting cycles.
Figure 3 Histological criteria used for molt staging. The cuticles of the black tiger shrimps in
different molting stages were routinely processed for histological study and stained with PAS
and Hematoxylin (a, c-h) or modified Masson’s trichrome (b). Each cuticular layer is
indicated in numbers 1-6. All figures are with the same magnification.
Figure 4 Epidermal morphology throughout the molting cycle. Intact cuticular tissue from the
trunk in various molting stages were routinely processed for histological study and stained
with modified Masson’s trichrome. C, cuticle; E, epidermis. All figures are with the same
magnification.
Figure 5 Carbohydrate deposition in the sub-epidermis throughout the molting cycle.
Cuticular tissues in each stage were routinely processed for histological study and stained with
either PAS and Hematoxylin (a-f), or toluidine blue (g-h). E, epidermis; Ga, tegumental gland
type A; Gb, tegumental gland type B; SE, sub-epidermal cell; Mu, mucus; N, nucleus; ED,
excretory duct; arrow = intercellular boundaries, * = metachromasia reaction.
Figure 6 Protein deposition in the sub-epidermis throughout the molting cycle. Cuticular
tissues in each stage were routinely processed for histological study and stained with modified
trichrome. E, epidermis; SE, sub-epidermal cell. All figures are with the same magnification.
Figure 7 Proteins in epidermis and sub-epidermis throughout the molting cycle.
Protein extract of the epidermis and sub-epidermis from a shrimp at each molting stage (D0 –
C) was subjected to 10% SDS-PAGE and stained with silver nitrate (a). Lane 1 (M) = protein
marker. Arrows indicate protein bands (1-8) being densitometry and statistical analysis.
Differences among means were analyzed from triplicate experiments by one way ANOVA
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followed with Turkey’HSD test. Figure 7b presents mean and S.D. of band 2 at each molting
stage. Statistical differences between D2, D3, D4, or A versus B and D2, D3, D4, or A versus
C are indicated (*, P < 0.05). The percentage of total intensity at each molting stage was also
indicated on the top of each histogram.
Figure 8 Peptide mass fingerprint of the 57 kDa protein band. The 57 kDa band was cut from
the Coomassie-stained gel, and de-stained. The protein was next in-gel digested with an
enzyme Trypsin, and the mass spectrums were analyzed with MALDI-TOF mass spectrometer
(Reflect IV, Bruker).
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S
D0 stage
D1 stage D2 stage
D3 stage
A stage
D4 stage
B-C stage
SC
C stage
a b
c d
e f
g h
EE
**
L* L*Sn
SCn
E
S
E
S
E
S
E
S
E
S
EI
EE
SnSn
Figure 1
50 µm
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Premolt (D0-4) 6-7 days
Intermolt (C3-4) 1-2 days
Postmolt (A1-2, B1-2, C1-2) 2-3 days
Ecdysis (E) Few minutes
Figure 2
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D2 stage D3 stage
B1 stage B2 stage
A1 stage
C 3-4 stage
1 2 3 4
1
2
3
1
2
1
2
3
1
2
3 - 4
5
1
2
3 - 4
c d
f
g h
a
10 µm
D4 stage
2
6e
5 3 - 4
1
Figure 3
C 3-4 stage
b
1 2 3 4
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E
E
E
E
E
b
c d
e f
D2 stage D3 stage
D1 stage
A stage B stage
C
C
C
CC
E
a
10 µm
C 3-4 stage
C
Figure 4
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D2 stage
A stage B stage
D0 stage
E
E
SE
E
Gb
Gb
SE SE
b
d
e f
Ga
C 3-4 stage
a
h 15 µm
Mu*
g 10 µm
10 µm 10 µm
10 µm
10 µm 10 µm
ED
N
N
Figure 5
D1 stage
SE
10 µm c
E
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D2 stage
A stage B stage
D0 stage
E
E
SE
E
SE
SE
b
d
e f
C 3-4 stage
E
a
10 µm
Figure 6
D1 stage
SE
E
c
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Sub-epidermal contents Molting stages Physical features of tegument Observed
cuticular layer Epidermal cell
morphology PAS positive cell
Ponceau S positive cell
Tegumental glands
C mature SCs, fully spread epidermis Ep, Ex, En, Ml short, untidy, loose - - -
D0 EE at base of SCs - - -
D1 narrow space between SCs and EE
short, tidy, dense + + +
D2 space wider straight EE
Ep, Ex, En
++ ++ +
D3 space wider, zig-zaged EE Ep, Ex, Ed, new Ep +++ +++ ++
D4 space widest, dense and zig-zaged EE, deformed SCs Ep, Ex, new Ep, new Ex ++++ ++++ ++
A young setae, no observed SCs new Ep, new Ex
very tall, tidy, dense
+++ +++ ++
B different sizes of SCs new Ep, new Ex, new Ed tall, tidy, dense ++ ++ +
Table 1 Summary of physical features of integument, observed cuticular layer, epidermal cell morphology, and sub-epidermal contents
corresponding to each molting stage. EE = epidermal edge, En = Endocuticle layer, Ep = Epicuticle layer, Ex = Exocuticle layer, Ml =
Membranous layer, SCs = setal cones. Ability to detect is represented by +, ++, +++, and ++++, respectively. - = not observed.
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Figure 7
M D0 D1 D2 D3 D4 A B C 105
75 50
35
30
25
15
10
(kDa)
a
12
34
56
78
% intensity
0 2 4 6 8 1
1
* *
b
Den
sito
met
ry
Molting stages D0 D1 D2 D3 D4 A B C
Fold increase 8.56 11.
72 17.74
17.11
18.63
20.68
4.20
1.97 4.
3 5.9 9.0 8.7 9.5 10.
5 2.1
1.0
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1203.412
1476.384
1975.510
1769.445
2438.550
1065.357
2210.5532593.695
1344.505
3297.360
1017.383
1642.410
2806.747
0
2
4
6
8
4x10In
tens
. [a.u
.]
1000 1250 1500 1750 2000 2250 2500 2750 3000 3250m/z
Figure 8