アサヒガニの幼生の成長にともなう消化酵素活性の変化

誌名誌名 水産増殖 = The aquiculture

ISSNISSN 03714217

巻/号巻/号 594

掲載ページ掲載ページ p. 521-528

発行年月発行年月 2011年12月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Aquaculture Sci. 59 (4) , 521 - 528 (2011)

Changes in Digestive Enzyme Activities of Larval and Early Crab Stages of

Red Frog Crab Ranina ranina with Growth

Yuko KUROKAWA1,*, Shin'ichiro KAWAI2, Kenzo YOSEDA3, Kazuhisa HAMADA4

,

Masaru TANAKA5 and Masashi .ANDol

Abstract: Developmental changes in digestive enzyme activities from zoeal to early crab stages in red frog crab (Ran ina ranina) were investigated in the laboratory. Four thousands larvae were kept in 0 .5 m3 tank at the temperature in ambient conditions (26.3 ± 1.6t). They were fed on Artemia nauplii from the first to fourth zoeal stages, thereafter frozen Japanese littlenecks, mysids, krills, and copepods were also given till early crab stage according to the developmental stages. The activities of trypsin-like enzyme and amylase were measured from one to 54 days after hatch­ing (DAH). Both enzymes were clearly detected from the first zoeal stage (0 DAH), and they were elevated with growth, especially from the fifth zoeal stage (16 DAH). A marked increase of body weight was also observed from 16 DAH, and it was well synchronized with increases of digestive enzyme activities. Differentiation of functional midgut gland was clearly observed at 16 DAH sug­gesting that fundamental structure of the digestive systems were differentiated at this stage. The increase of trypsin-like enzyme and amylase activities coincided well with the differentiation and the development of midgut gland based on histological observation.

Key words: Red frog crab; Midgut gland; Trypsin-like enzyme; Histological observation

Red frog crab (Ranina ranina) is widely distributed in the temperate to the subtropi­cal zone in the Pacific and Indian Oceans, and this crab is delicious, valuable and commer­cially important in Kagoshima and Miyazaki prefecture in Japan (Miyake 1983). The catch of this crab has been decreasing reacentiy, therefore, the development of seed produc­tion and the release technique of this crab has been expected. Because of the lower sur­vival rate in their larval stages, mass scale production of juveniles is now unsuccessful in the several experimental research stations

Received 7 March 2011; Accepted 4 July 2011.

(Ashidate 1988; Minagawa and Kudo 1988; Kagoshima Prefectural Fisheries Technology and Development Center 1992) . To exploit the proper dietary organisms is necessary for the development of seed production of this crab.

In order to develope stable rearing techniques, their biological knowledges related with rear­ing environment conditions such as water tem­perature, salinity, light condition, larval stocking density, and food quality and quantity are needed to investigate. Studies on digestive physiology during larval and early crab stages are also nec­essary to elucidate their digestive ability.

1 Department of Fisheries, Faculty of Agriculture, Kinki University, 3327-204, Nakamachi, Nara, Nara 631-8505, Japan. 2 Department of Food Design, Faculty of Nutrition, Koshien University, 10-1, Momijigaoka, Takarazuka, Hyogo 665-0006, Japan. :3 National Research Institute of Fisheries and Environmental of Inland Sea, Fisheries Research Agency, 2-17-5, Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan. 4 Komame Station, Stock Enhancement Technology Development Center, National Research Institute of Aquaculture, Fisheries Research Agency, Komame, Ootsuki, Kochi 788-0315, Japan. S International Institute for Advanced Studies, 9-3, Kizugawadai, Kizugawa, Kyoto 619-0225, Japan. * Corresponding author: Tel, (+81) 742-43-6299; Fax, (+81) 742-43-1316; E-mail, yuko-k@kvf.biglobe.ne.jp 0/. Kurokawa).

522 Y. Kurokawa, S. Kawai, K. Yoseda, K. Hamada, M. Tanaka and M. Ando

Many studies on digestive enzymes of the crustaceans have been actively conducted in the past. These studies mainly focus on the dif­ference of activities among crustacean species, organ-localization of the digestive enzymes, properties and purification of their digestive enzymes. As for properties of digestive enzymes of the crustaceans, various kinds of enzymes including trypsin (Devillez and Buschlen 1967; Dendinger 1987; Galgani and Nagayama 1987), trypsin-like enzyme (Dittrich 1990), chymotryp­sin (Dendinger 1987), aminopeptidase (Omondi and Stark 2001), carboxypeptidase A and B (Dendinger 1987; Galgani and Nagayama 1987), elastase (Dendinger 1987), collagenase (Galgani and Nagayama 1987), and amylase (Sather 1969) were investigated. However, most parts of these studies focus on adult stage and very few reports have been made on the development of digestive enzyme activities during larval and early crab stages with growth.

The various studies of red frog crab were conducted on the growth (Minagawa and Kudo 1988), developmental changes in body weight and elemental composition of laboratory-reared larvae (Minagawa et a1. 1993), effects of prey den­sity on survival, feeding rate and development of zoeas (Minagawa and Murano 1993a), larval feed­ing rhythms and food consumption (Minagawa and Murano 1993b), developmental changes in larval mouthparts and foregut (Minagawa and Takashima 1994). However, the relationship between changes in digestive enzyme activities and development of digestive system with the larval growth has not been studied.

In this paper, we investigated development of digestive enzyme activities and digestive system with growth of the species to obtain important bio­logical knowledges on their digestive physiology.

Materials and Methods

Broodstock and larval managements The parent crabs were caught by a kind of

gill net at the coast of Kagoshima and Miyazaki prefecture in 1994, and only the female with eggs were transported to Shibushi Station, National Center for Stock Enhancement, Fisheries

Research Agency, and they were kept in double bottom tank (FRP tank, 1.6 m3) laid with sand between 1 and 35 days after stocking. Thereafter, each female with eggs just before hatching were individually selected, and they were kept under a static water condition with weak aeration (0.3 mllmin) in another black polyethylene tanks (0.5 m3). Four thousands of hatched larvae from one matured female crab were reared in a tank. Seawater filtered with sand and mechanical fiber filter (0.2 f.1 m) was used as the rearing water.

Four thousands red frog crab larvae after hatching were transported to experimental polyethylene tanks (0.5 m3) using a pipette. The microfiltrated seawater was used as rearing water of the experiment, and the water temperature was adjusted to 26.3 ± 1.6'C . The larvae were fed on Artemia nauplii (Artemia salina) from the first to the fourth zoeal stages, thereafter frozen Japanese littlenecks, mysids, hills, and frozen copepods were also given till early crab stage (Table 1). The density of Artemia in rearing sea­water was kept one individual per ml. The rearing water was completely changed at once a time per day, thereafter the larvae were transferred using a pipette to new tank every day.

Sampling

Larval and early crab stages samples were collected in each stage during the experimen­tal period between July 7 and September 8 in 1994. The molting stage was classified based on microscopic observation of larval morphol­ogy and the number of exuviae at the bottom in the experimental tank. Sampling of larvae in

Table 1. Diet of red frog crab in developmental stage

Stage Feed

Zoea I-IV stage

Zoe a V-VII stage

Megalopa and early crab stage

Artemia nauplii (Artemia salina) Formula feeds

Artemia nauplii (Artemia salina) Formula feeds Frozen Japanese littlenecks Mysids Krills Copepocls

Frozen Japanese littlenecks Mysic1s Krills Squids

Digestive Enzyme Activities of Red Frog Crab 523

each stage was conducted when more than 80% of larvae completed exuviations. The samples being 0.2 g to 1.8 g of wet weight were imme­diately transferred to collection bag (Unipack mark-D, Seisannipponsha Ltd, Tokyo, Japan) and were kept at - 80°C in a deep freezer (U581, SANYO Electric Co., Ltd, Osaka, Japan) until the digestive enzyme assay. To conduct histological observation in larval and early crab stages, ten samples were fIxed by soaking in Bouin's solu­tion for three hours and preserved in 80 - 90% ethanol, then dehydrated in ethanol till 100%.

Preparation of crude enzyme solution The number of larvae and juveniles used for

the enzyme assay in each two group were 500 in Zoea I stage, 300 in Zoea II stage, 100 in Zoea III stage, 50 in Zoea IV stage, 30 in Zoea V stage, 3 in megalopa stage and 2 in early crab stage. One group larvae was used for the enzyme assay in Zoea VI and Zoea VII stages, and the number of larvae were 20 and 10, respectively. Each sample preserved at - 80°C in a freezer was moved to 10 ml stoppered test tube and shredded into small pieces with dissection scissors keeping cold with ice. Seven ml of 1/15 M phosphate buffer (PH 7.0) was added to the cut sample and the sample was homogenized for 3 min with an electric homogenizer. After centrifuging the homogenate at 10,000 x g for 20 min and fIltering the supernatant with silica wool, the phosphate buffer (pH 7.0) was added to the fIltrate to make it 10 ml and was used as a crude enzyme solution (Kawai and Ikeda 1971).

Measurement of digestive enzyme activities The tryptic activity was assayed using 3%

of milk casein solution as a substrate and free aromatic amino acid liberated was measured spectrophotometrically by the Folin method (Kawai and Ikeda 1972, 1973a). Amylase activity was assayed using 1 % of soluble starch solution as a substrate and liberated reducing sugar was determined by Somogyi-Nelson method (Kawai and Ikeda 1972, 1973a).

To measure enzyme activities, a crude enzyme solution, buffer solution and substrate solution were added to test tubes and was incubated for

30 min at 37°C with shaking. The enzyme reac­tion was stopped by adding trichloroacetic acid for trypsin-like enzyme activity, and by boiling for 5 min for amylase activity. Thereafter, a quan­tity of reaction product was determined by each method and the activities were expressed as total activity (f-J- g of tyrosine or glucose liberated 1 30 min 1 individual) and activities per weight (f-J- g of tyrosine or glucose liberated 1 30 min 1 mg of body weight) (Kawai and Ikeda 1973b).

Histological observation Fixed samples with Bouin's solution were

embedded in parahisto (paraffIn for histology, Nakarai) after immersion in xylene (Tanaka et al. 1996). The samples embedded in para­histo were sectioned at 4 f-J- m thickness using a microtome (Large Rotary Microtome LR-85, Yamato Kohki Industrial Co., Ltd., Saitama, Japan) and placed on slide glasses. After dis­solving parahisto, the samples were double stained with hematoxylin solution and eosinl erythrosine solution. They were enclosed in a cover glass using Canada balsam and were observed with an optical microscope.

Statistics Pearson's correlation coeffIcient test was

used to analyze the correlation between trypsin­like enzyme and amylase activities at 5% sig­nifIcance level using the STATCEL2 program (4 steps Excel Toukei, OMS Publishing Inc., Saitama, Japan), an add-in to the Excel software (Excel 2002, Microsoft Corp., Tokyo, Japan).

Results

Growth The average weight ranged from 0.793 to

50.5 mg during zoeal stages, and it was 176 mg and was 298 mg at megalopa and early crab stages, respectively (Fig. 1). Marked weight gain observed from the fIfth to the seventh zoeal stage and from the seventh zoeal to early crab stages.

Changes in Trypsin-like enzyme activities Though the weak total activity of just hatched

zoeal larva (Zoea I stage, 0 DAB) was clearly

524 Y. Kurokawa, S. Kawai, K. Yoseda, K. Hamada, M. Tanaka and M. Ando

detected, it showed almost the same level till 8 DAB (Zoea III stage). Then it was gradually increased at 12 DAB (Zoea IV stage), thereafter marked increase was detected during 16 DAB (Zoea V stage) and 25 DAB (Zoea VTI stage) same as body weight gain mentioned above (Fig.2A).

Regarding the activity in body weight basis, it showed the up-and-down aspect repeatedly according to developmental stages. It reached the maximum level around at 16 DAB (Zoea V stage) and decreased afterwards (Fig. 2B).

M .s E b.O 'iii ;: >-'0 0 aJ

350

300

250

200

150

100

50

o 10 20 30 40 50 60

Days after hatching

Fig. 1. Growth of red frog crab after hatching. Average body weight of each stage obtained from 10 to 100 larvae or juveniles in each two group (.) and its mean (e) are shown in the figure. Zl - Z7, first to seven zoeal stages; M, megalopa stage; C, early crab stage.

1,000 A

~ 900 'C

'" 800 '6 ,,= , 700 ~.£:

+" E 0:;: 0

.- '" 600 1:5' rnal 500 -<ii rn ~

400 o@ I- " 300 ,,=

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0 0 10 20 30 40 50

Days after hatching 60

(f)

Changes in Amylase activities The total activity at 0 DAB (Zoea I stage) was

weak and showed almost the same level till 8 DAB (Zoea III stage). Thereafter the activity at 16 DAB (Zoea V stage) was increased one point five times higher than that at 8 DAB (Zoea III stage) (Fig. 3A).

Regarding the activity in body weight basis, it showed the up-and-down aspect same as trypsin-like enzyme during zoeal and megalopa stages, and finally slightly increased at early crab stage (Fig. 3B).

The variation pattern of trypsin-like enzyme and amylase activities exhibited positive cor­relation by Pearson's correlation coefficient test (r = 0.94, P < 0.05).

Development and differentiation of digestive

system with growth

Foregut, midgut and midgut gland of larvae were clearly differentiated immediately after hatching (Zoea I stage), but no differentiation was observed for the anterior and posterior foregut (Fig. 4A).

A constriction was appeared between the ante­rior and posterior foregut at 16 DAB (Zoea V stage). A small teeth-shaped or comb-like denticle structure was observed on the ventral surface of the posterior foregut at 16 DAB (Zoea V stage). In the anterior foregut, the shells of Artemia nau­plii were found to be crushed. The tufted midgut gland started to be differentiated at 16 DAB (Zoea

E 12 bIl 'ij; B ~

'~ -g 10 .c:;:

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0 10 20 30 40 50 60 Days after hatching

Fig. 2. Development of trypsin-like enzyme activity of red frog crab with growth. Activities of trypsin-like enzyme are expressed as total activity (A) and activity in body weight basis (B), respectively. Activity of each two group (.) and the mean (e) were shown in the figure. Zl - Z7, first to seven zoeal stages; M, megalopa stage; C, early crab stage.

Digestive Enzyme Activities of Red Frog Crab 525

1,200

~ " 1,000 .>

~ , :>. .~ .~ E 800 > 0

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U ' 600 CU " ..

(lj ~ - .. o ~ ~ ~ 400

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1 0 20 30 40 50 60 0 1 0 20 30 40 50 60

Days after hatching Days after hatching

Fig. 3. Development of amylase activity of reel frog crab with growth. Amylase activities are expressed as total activity (A) and activi ty in body weight basis (B), respectively. The number of larvae and juveniles used for enzyme assay are same as the description in Fig. 2 .

..---1 ," r" ., 7

• ----- -< -- me ... 41 6

. "":;.

A J

1~m IIIIIi!'

;

~.

r--~

cp

Fig. 4. Development of digestive organ of red frog crab with growth. A, first lOeal stage; B, fifth zoeal stage; C, megalopa stage; D, early crab stage. cp, cardiac por tion of proventriculus; e, esophagus; h, hindgut; m, midgut; me, midgut caecum; mg, midgut gland; pp, pyloric portion of proventriculus; pr, proventriculus.

V stage), and its total volume and inside area has markedly enlarged at this stage. Though midgut gland seems tufted, basic portion of the gland is formed one tube and is opened to the anterior part of midgut. There was also a midgut caecum behind the start site of midgut at 16 DAR (Zoea V stage). At this stage, the basic structure of diges­tive system was functionally completed (Fig. 4B).

Thirty-seven days after hatching (megalopa stage), the anterior foregut was enlarged fur­ther and its interior surface was covered with a cuticular membrane and showed complex features. The septal wall of the anterior foregut

(cardiac part) and posterior foregut (pyloric part) was developed further and showed sev­eral small teeth on the inner surface. The inner cavity of midgut was occupied with pieces of food which were crushed in the anterior fore­gut. Some tissue fragments which were con­sidered to be deciduous cell clusters of walls of digestive tract were also observed in the midgut lumen, suggesting that the tufted structure of midgut gland developed further (Fig. 4 C).

The posterior foregut was contracted because we used sample organisms which had not fed, however the both dorsal and ventral sides were

526 Y. Kurakawa, S. Kawai, K. Yaseda, K. Hamada, M. Tanaka and M. Anda

covered with teeth-like structures at 54 DAH (early crab stage). The midgut gland developed further and branched into tufts or areolars show­ing the clear increase of both surface area and volume. Many balloon-like structures which were regarded as absorptive cells of midgut gland, were also observed, indicating that the digestive and absorptive function reached at almost complete level at this stage (Fig. 4D).

Discussion

The total activity of typsin-like enzyme remained at low level from just hatched larva (Zoea I stage) to 8 DAH (Zoea III stage). However, the total activity level gradually increased after 12 DAH (Zoea N stage) and showed marked increase around at 16 DAH (Zoea V stage). Changes in the digestive enzyme activities were considered to be related to the functional development of digestive organ which began around at 16 DAH (Zoea V stage). Additionally, marked increase of body weight gain coincided with that of the total activitiy. So far, we have investigated on digestive enzyme activities in the early life stages of approximately 30 species of fish (Kawai and Ikeda 1971, 1972, 1973a, 1973b; Tanaka et al. 1996; Kawai 2001; Yoseda et al. 2003a, 2003b; Fujii et al. 2007, Teruya et al. 2008) to clarify their development in relation to those of digestive system. As the result of the study, it was demonstrated that the total activity of enzymes increased especially after the period of marked weight gain (Kawai 2001). In this study, we could also demonstrate almost the same tendency for a crustacean as various kinds of fishes including flat fishes, seliora species, flying fish, etc.

On the other hand, the trypsin-like enzyme and amylase activities in body weight basis reached maximum level at the period of marked weight gain, and after that either an equilibrium state or a downward tendency was observed. Based on these results we concluded that the period of marked weight gain might be turning point of food. The midgut gland, being a major organ in the digestive system was in the almost completely differentiated functionally around at 16 DAH (Zoea V stage). The digestive enzyme

activities per weight reached the highest level around at 20 to 30 DAH when the develop­ment of digestive system almost completed functionally in many kinds of fish species (Kawai 2001), and in fact, diet switching is commonly conducted around this period in seed production.

The correlation coefficient between the develop­ment of trypsin-like enzyme and amylase activi­ties and that of body weight were 0.951 and 0.984, respectively, from the statistic analysis by Pearson' s product-moment correlation coefficient.

Additionally, the activity of trypsin-like enzyme measured using casein as a substrate, was confirmed to be trypsin itself by using a synthetic substrate, Na-benzoyl-L-arginine methylcoumaryl­amide.

Significance of existence of amylase in carniv­orous organisms remains still unknown, though amylase surely exists in the early life stages of fish and also in larval stages of crustaceans. It requires further researches to clarify the mean­ing, although there is a possibility of the hydro­lysis of glycogen pooled in the body.

As shown in Fig. 4A to 4D, a clear differen­tiation of foregut, midgut and midgut gland was observed on the day of hatching (Zoea I stage), however, the differentiation of the ante­rior foregut (cardiac part) and posterior foregut (pyloric part) were not visible and were still in a globular shape, indicating that the functional digestive system was low at this stage. This is consistent with the report that the proventricu­lus of instar I zoea was small and the median tooth was poorly developed (Minagawa and Takashima 1994). The midgut gland was not observed to be tufted and was simply like a bag. There were extremely limited numbers of cells such as absorptive cells, fiber cells and vacuolar cells which are responsible for degradation of nutrition taken in vacuoles (Dall and Moriatry 1983; Al-Mohanna and Nott 1986), meaning low trypsin-like enzyme and amylase activities.

The digestive system also started to develop progressively from zoeal stages. The secretory function of the digestive organ seemed to com­plete on 16 DAH (Zoea V stage). In facts, the activities of trypsin-like enzyme and amylase showed a rapid increase during this period and

Digestive Enzyme Activities of Red Frog Crab 527

a clear coincidence was confirmed from the evi­dences of digestive physiology and morphology based on a histological method. There is also a clear differentiation of midgut caecum from the anterior midgut, however, its main function is unknown, being assumed nearly the same role as the midgut (Dall and Moriatry 1983).

As shown in Fig. 4 C, the ability to crush the ingested food physically was increased because several small teeth were observed on the inner surface of the foregut. The further development of the tufted structure of midgut gland coincides well with the clear increase of trypsin-like enzyme and amylase activities during this period.

Fifty-four days after hatching (early crab stage), the tufted structure of midgut gland developed significantly and differentiated into numerous tufts with the further increase of trypsin-like enzyme and amylase activities compared to the larvae of 37 DAB (megalopa stage).

In conclusion, the result of this study shows that the increase of trypsin-like enzyme amylase activities, and body weight gain coincide well with the developmental state of the midgut gland at 16 DAB (Zoea V stage). It means that the fifth zoeal stage might be occurred endogenous and/or exogenous changes in accordance with growth and digestive physiology. Additionally, it is also important to obtain more knowledge about diges­tive physiology in various kins of marine crus­taceans. The assay method of digestive enzyme activities used in this study required 0.3 to 0.5 g of sample in wet weight and hundreds and more individuals of larvae were necessary when the body weight is below 1 mg. Recently, a precise method to measure enzyme activities with only single fish larva has developed, and activities of enzymes such as trypsin, chymotrypsin, amylase and lipase for some kinds of fish larva have been reported using this method (Ueberschar et al. 1992; Ozeki and Biley 1995; Fujii et al. 2007).

Following researches are essential including relationship between health condition of larvae and the digestive enzyme activities, diurnal variation of the digestive enzyme activities, and various environmental factors such as water temperature, salinity, dissolved oxygen, light­ing, etc. as well as biotic factors like stocking

density and feeding incidence, which are responsible for the digestive enzyme activities.

Acknowledgements

We are grateful to the staff of Shibushi Station, National Center for Stock Enhancement, Fisheries Research Agency for their help in rearing and sampling of red frog crab larvae and early juveniles, and to Ms. Yasuko Kaji for her help in preparing histological sections. This work was supported by Kinki University Global COE Program for International Education and Research Center for Aquaculture Science of Bluefin Tuna and other Cultured Fish. We would like to express our sincere thanks to all the per­sons concerned.

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Yoseda, K, S. Dan, A Fujii, Y. Kurokawa and S. Kawai (2003b) Effects of different photo periods on first­"feeeling succecss, early survival and digestive enzyme activities in coral trout Plectropomus leopardus larvae. Suisanzoshoku, 51,179-188 (in Japanese).

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