Comparative Biochemistry and Physiology€¦ · ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Promwikorn...

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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

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http://dx.doi.org/10.1016/j.cbpb.2007.04.009

http://dx.doi.org/10.1016/j.cbpb.2007.04.009

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ACCEPTED MANUSCRIPTPromwikorn et al., 2007

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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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ACCEPTED MANUSCRIPT

Promwikorn et al., 2007

24

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

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