Expression of Pancreatic Enzyme Genes During the Early Larval Stage of Japanese Eel Anguilla Japonica

FISHERIES SCIENCE 2002; 68: 736–744

Expression of pancreatic enzyme genes during the early larval stage of Japanese eel Anguilla japonica

Tadahide KUROKAWA,* Tohru SUZUKI, Hiromi OHTA, Hirohiko KAGAWA, Hideki TANAKA AND Tatsuya UNUMA

National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 516-0193, Japan

ABSTRACT: To reveal the ontogeny of pancreatic exocrine function in the early larval stage of eel, cDNAs encoding major pancreatic enzymes, trypsinogen, amylase and lipase were identified from the Japanese eel Anguilla japonica and their expression pattern in larvae was analyzed. The cloned eel trypsinogen precursor consisted of 224 amino acids and showed 82.2% identity to trypsinogen- 2 of winter flounder Pleuronectes americanus. The eel amylase precursor consisted of 512 amino acids and showed 77% identity to winter flounder amylase. Eel pancreatic lipase was composed of 470 amino acids and had 58.3% of identity to human pancreatic lipase. In the eel larvae, mRNA expression of trypsinogen and amylase was first detected at 6 days post-hatching (d.p.h.), and the expression level increased between 7 and 8 d.p.h. In contrast, mRNA expression of lipase was first detected at 8 d.p.h. Eel larvae start to feed actively at 8 d.p.h. Thus, it was indicated that eel pancreas starts to synthesize digestive enzymes at 6 d.p.h. and acquires full function by the onset of exogenous feeding at 8 d.p.h.

KEY WORDS: amylase, cDNA, eel, larvae, lipase, mRNA expression, pancreatic enzymes, trypsinogen.

INTRODUCTION digestive tract of wild Anguilliformes leptocephali feces of zooplankton and larvacean house were Anguilliformes, including eel, have a unique larval found.8 However, the natural food of eel prelepto- stage called leptocephalus.1 Tsukamoto2 estimated cephali is still unknown. Therefore, studies of the that the spawning area of the Japanese eel Anguilla digestive physiology in the larval stage are very japonica was west of the Mariana Islands where important for the development of a suitable diet many young leptocephali were collected. However, and culture systems for eel larvae. wild preleptocephali and eggs of Japanese eel have The pancreas is a very important organ for not yet been found. digestion, particularly in the larval stage in which Under experimental conditions, Yamamoto and the stomach is not yet developed.9 In most teleosts, Yamauchi3 first succeeded in producing Japanese it is difficult to collect pure pancreas because the eel larvae (preleptocephalus). In spite of many pancreatic tissue is diffusively distributed.10,11 Eel researchers’ efforts, there has been only one suc- is one of the few teleosts possessing a compact cessful report on growing larvae to the lepto- exocrine pancreas; therefore, the eel is adequate cephalus stage.4 Fertilized eggs and hatched larvae for the biochemical characterization of the pan- have also been obtained in the European eel creatic enzymes.12,13 However, when analyzing the Anguilla anguilla5,6 and the New Zealand eel synthetic ability of digestive enzymes in larvae, Anguilla dieffenbachii,7 but rearing larvae was gene expression analysis is more specific than unsuccessful. biochemical detection because some enzymes, A lack of information about the diet of larvae is which have the same activity as pancreatic probably one of the major reasons for their short enzymes, exist in other organs. In the present survival period in experimental conditions. In the study, we cloned cDNAs encoding the major pancreatic enzymes, trypsinogen, amylase and lipase, from the Japanese eel and analyzed their *Corresponding author: Tel: 81-599-66-1830. Fax: 81-599-66- expression patterns during the larval stage in order 1962. Email: [email protected] to reveal the development of enzyme synthetic Received 7 September 2001. Accepted 17 December 2001. ability in the eel larval pancreas. Pancreatic enzyme genes of eel FISHERIES SCIENCE 737

MATERIALS AND METHODS PCR products of the expected length for trypsinogen (530 bp), amylase (650 bp) and lipase (540 bp) Polymerase chain reaction amplification were purified from agarose gel and cloned into the pCRII vector (Invitrogen, Carlsbad, CA, USA). The pancreas (0.1 g) of Japanese eel Anguilla Inserts were sequenced using the Big Dye Termi- japonica (38 cm in total length, 2-year-old male) nator Cycle Sequencing FS Ready Reaction Kit was dissected and poly (A) + RNA was prepared (Applied Biosystems, Foster City, CA, USA) and using the QuickPrep mRNA Purification Kit an automated DNA sequencer (377 A; Applied (Amersham Pharmacia Biotech, Piscataway, NJ, Biosystems). USA). First-strand cDNA linked with 5¢- and 3¢- adapters for rapid amplification for cDNA ends (RACE) was synthesized using the SMART PCR cDNA amplification by 5¢- and 3¢-rapid cDNA Library Construction Kit (Clontech, Palo amplification for cDNA ends Alto, CA, USA). The primary polymerase chain reaction (PCR) Rapid amplification for cDNA ends PCRs (5¢- and primers (Table 1) were designed at the conserved 3¢-) were performed using primers designed in region of vertebrate trypsinogen (winter flounder, the adapter sequences of the SMART PCR cDNA GenBank AAC32752; Japanese flounder, BAA82362; Library Construction Kit and first and second sea bass, CAA07315), amylase (winter flounder, gene-specific primers designed in the cloned PCR AF252633; mouse, P00688) and pancreatic lipase fragments (Table 2), as previously described.14 The (mouse, A34671; human, C43357; rabbit, A28997). PCR parameters were 30 cycles of 95°C for 30 s, The cDNA of pancreas was used as a template for 58°C for 1 min, and 72°C for 2 min both for first and the primary PCR. Polymerase chain reactions were second PCRs. The RACE PCR products were puri- performed using primer sets, Try5¢ and 3¢ for fied from the gel, cloned into the pCRII vector and trypsinogen, Amy5¢ and 3¢ for amylase and Lip5¢ sequenced. and 3¢ for lipase, with Taq polymerase and an additional buffer (Takara, Ohtsu, Japan). The PCR parameters for trypsinogen were 30 cycles of 96°C Reverse transcription-PCR assay in larvae for 30 s, 50°C for 1 min, and 72°C for 2 min The parameters for amylase and lipase were 50 cycles Fertilized eggs of the Japanese eel were kept in a of 96°C for 30 s, 42°C for 1 min and 72°C for 2 min. 50 L tank supplied with running seawater (21 ± 1°C)

Table 1 Primer sequences of primary polymerase chain reaction for trypsinogen (Try), amylase (Amy) and lipase (Lip) 5¢ Primer 3¢ Primer Try5¢-CAYCAGGTGTCTCTGAAC Try3¢-CCCARGACACAACACCCTG Amy5¢-GCNTGYAARCAYATGTGGCC Amy3¢-SWYTGRTTRTCCCACCAGTT Lip5¢-AAYTGYATHTGYGTNGAYTGG Lip3¢-AWRTCNGCRTAYTTRTA

Table 2 Primer sequences of rapid ampliﬁcation for cDNA ends–polymerase chain reaction 5¢RACE 3¢RACE RACE adapter primer RACE5¢a-GCAGTGGTATCAACGCAGAGTGGCCA RACE3¢a-TAGAGGCCGAGGCGGCCGACAT RACE5¢b-GGTATCAACGCAGAGTGGCCATTACG RACE3¢b-AGGCCGAGGCGGCCGACATGTT Gene-speciﬁc primer Try5¢a-GAGTTTGAACAGTCGCTCTCC Try3¢a-TTCATCGGCGCTTCCCACGTC Try5¢b-ACCCAGGCGCACTTCCAAACGGCTC Try3¢b-TGGAAATCCCCATCCTGTCGGAGAG Amy5¢a-GGGCCAGCATAAAACCTGTGG Amy3¢a-TACTGGAATTGGTCGGGTCAC Amy5¢b-GCTGGACACAGCCTGCAGGTC Amy3¢b-GGTCATTTTCCGTAATGTTGTC Lip5¢a-AAGATGGCCGACAGCTTGAGC Lip3¢a-CCAGCAGAGGCCCGAGTCGGT Lip5¢b-GTACAGCGTCCGGCCGCCCTT Lip3¢b-GGCCTGCAACCATCTGAGGTC 738 FISHERIES SCIENCE T Kurokawa et al.

circulating at a flow rate of 1.2 L/min. Larvae RESULTS hatched 2 days post fertilization. From 7 days post- hatching (d.p.h.), larvae were kept in 5 L acrylic Cloning of eel pancreatic enzyme cDNA resin bowl tanks and reared by the culture system developed by Tanaka et al.4 using Aquaran (BASF, Sequence analysis indicated that trypsinogen Tokyo, Japan) as the diet. (544 bp), amylase (653 bp) and lipase (518 bp) Messenger RNA of whole larvae was prepared cDNA fragments were amplified from the eel pan- at 0, 1, 3, 4, 6, 7, 8, 9 and 10 d.p.h. (five individuals creas by PCR using degenerated primers (Table 1). were pooled at each stage) using the QuickPrep The full sequences of eel trypsiogen, amylase and mRNA Purification Kit (Amersham Pharmacia lipase cDNA were successfully obtained by 5¢- and Biotech) and cDNA was synthesized using First- 3¢-RACE amplifications using primers designed strand cDNA Synthesis Kit (Amersham Pharmacia from the sequences of the cloned PCR fragments. Biotech). The eel trypsinogen precursor (GenBank Polymerase chain reaction was performed using AB070720) that was obtained consisted of 244 primer sets for RT-PCR (Table 3). The PCR para- amino acids including a putative 15-amino-acid meters were 30 cycles of 95°C for 30 s, 60°C for signal peptide and five-amino-acid activation 1 min and 72°C for 2 min Eel b-actin was amplified peptide (Fig. 1). The eel trypsinogen has a high to confirm the steady-state level of expression of identity to winter flounder trypsinogen-2 (82.2%), the housekeeping gene. Japanese flounder trypsinogen-1 (78.1%) and Maori cod (76.9%). The eel amylase precursor (GenBank AB070721) In situ hybridization consisted of 512 amino acids. The position of the cleavage site for a signal peptide was conserved Antisense riboprobes were transcribed from after position 15 alanine, as in the case of winter the clones of primary PCR amplification for flounder and mouse amylase (Fig. 2). The eel trypsin, amylase and lipase using the Boehringer amylase showed a high identity to the amylase of Mannheim RNA Labeling Kit (Boehringer winter flounder (77%) and mouse (72.6%). Mannheim, Basel, Switzerland) with digoxigenin The eel lipase precursor (GenBank AB070722) (DIG)-UTP. Sense riboprobes were also tran- consisted of 470 amino acids including an 18- scribed as negative controls. Whole-mount In amino-acid signal peptide (Fig. 3). It showed a situ hybridization (ISH) was performed on fixed 58.3% identity to human pancreatic lipase, but a 10 d.p.h. larvae according to Jowett and Lettice.15 lower identity to human hepatic (26.7%) and

Table 3 Primer sequences for analysis of trypsinogen (Try), amylase (Amy) and lipase (Lip) expression 5¢ Primer 3¢ Primer Try1-TTCATCGGCGCTTCCCACGTC Try2-GAGTTTGAACAGTCGCTCTCC Amy1-TACTGGAATTGGTCGGGTCAC Amy2-GGGCCAGCATAAAACCTGTGG Lip1-CCAGCAGAGGCCCGAGTCGGT Lip2-AAGATGGCCGACAGCTTGAGC Act1-CTCCCTGGAGAAGAGCTACGAG Act2-ATCCAGACGGAGTATTTGCGCT

Primer position: Try1 (281–301 of AB070720 in GenBank), Try2 (520–540 of AB070720), Amy1 (811–831 of AB070721), Amy2 (1041–1061 of AB070720), Lip1 (481–501 of AB070722), Lip2 (713–733 of AB070722), Act1 (1–22 of AB074846), Act2 (300–321 of AB074846).

Fig. 1 Alignment (a) and phylogenetic tree (b) of amino acid sequences of eel trypsinogen and fish trypsinogen. GenBank accession numbers: winter flounder (Pleuronectes americanus)-1, AAC32751; -2, AAC32752; -3, AAC32753; Japanese flounder (Paralichthys olivaceus)-1, BAA82362; -3, BAA82364; Maori cod (Paranotothenia magellanica), CAA57701; Atlantic cod (Gadus morhus), S39047; plaice (Pleuronectes platessa), CAA40068. Deletions are indicated by dash. Amino acid residues common to eel trypsinogen are indicated by shaded areas. Arrows indicate the positions of cleavages of the signal peptide and activation peptide. Fish trypsinogen can be divided into three types according to winter flounder trypsinogen.16 Alignment was performed by the clustal W program (http://www.ddbj.nig.ac.jp). The phylogenetic tree was created by boostrap N-J tree program (http://www.ddbj.nig.ac.jp) (scale bar: substitutions per site). Pancreatic enzyme genes of eel FISHERIES SCIENCE 739 740 FISHERIES SCIENCE T Kurokawa et al.

Fig. 2 Alignment of amino acid sequences of amylase from eel, winter flounder (AAF65827), and mouse (P00688). Deletions are indicated by dash. Arrow indicates the predicted site of signal peptide cleavage. Alignment was performed by the clustal W program (http://www.ddbj.nig.ac.jp). lipoprotein lipase (26.5%) and red sea bream using the gene-specific primers indicated in Table lipoprotein lipase (25.9%). Until the present study, 2. Eel larvae hatched 2 days after fertilization, when a pancreatic lipase sequence had not been the mouth had not yet opened. PCR amplification reported from any fish. Because the eel lipase pre- derived from pancreatic enzyme was not detected cursor showed a greater identity in size and struc- at this stage. ture to human pancreatic lipase than to human Eel larvae started to feed actively at 8 d.p.h.. hepatic and lipoprotein lipase, we concluded that Messenger RNA expression of trypsinogen and this eel lipase is pancreatic lipase. amylase was first detected at 6 d.p.h. and the expression level increased between 7 and 8 d.p.h. (Fig. 4). After this time, lipase mRNA expression Tissue expressions of pancreatic enzymes started at 8 d.p.h., but the expression level was mRNA in larvae lower than that of trypsinogen and amylase even in the 10 d.p.h. larvae (Fig. 4). Messenger RNA expression of trypsinogen, In the whole-mount ISH analysis, the hybridiza- amylase and lipase precursor genes during the tion signals to the antisense probes for trypsino- early larval stage was analyzed by means of RT-PCR gen, amylase and lipase were observed only from Pancreatic enzyme genes of eel FISHERIES SCIENCE 741

Fig. 3 Alignment of amino acid sequences of eel pancreatic lipase (PL), human PL (GenBank accession number C43357), human hepatic lipase (HL; A28997), human lipoprotein lipase (LPL; P06858) and red sea bream LPL (BAB20996). Amino acid residues common to eel lipase are indicated by shaded areas. Arrow indicates the predicted site of signal peptide cleavage. Box indicates common sequences of lipase. Alignment was performed by the clustal W program (http://www.ddbj.nig.ac.jp). 742 FISHERIES SCIENCE T Kurokawa et al.

Fig. 4 Reverse transcription–polymerase chain reaction analysis of expression of pancreatic enzymes in eel larvae and adult pancreas. the pancreas of 10 d.p.h. eel larvae (Fig. 5). In contrast, the hybridization signals to sense probes were not detected. Therefore, the results of RT-PCR analysis indicated mRNA expression of digestive enzymes in the larval pancreas.

DISCUSSION

The cDNA of major pancreatic enzymes (trypsino- Fig. 5 Whole-mount in situ hybridization of 10 d.p.h. gen, amylase and lipase) was isolated from the eel larvae with antisense probes of pancreatic digestive Japanese eel pancreas to analyze the expression enzyme genes. (a) Trypsinogen, (b) amylase, (c) lipase. pattern of digestive enzymes in the larvae. Arrowheads indicate positive signals in the larval pan- Trypsinogen is a zymogen of trypsin, which is one creas. Scale bars, 1 mm. of the major pancreatic proteases. Douglas and Gallant16 reported that fish trypsinogen could be divided into three types according to the gene Scophthalmus maximus20 and yellowfin tuna analysis of winter flounder trypsinogen. As shown Thunnus albacares.21 Trypsinogen was first in Fig. 1, the eel trypsinogen isolated in the detected immunohistochemically in the pan- present study seems to belong to winter flounder creatic zymogen granules of Japanese flounder trypsinogen-2 type. This type of trypsinogen has by the first feeding22 and amylase activity was also been detected in a wide variety of teleosts.16 detected by this stage in striped bass Morone Amylase is a major digestive enzyme for carbo- saxatilis,23 and turbot.24 hydrates. With regard to fish amylase, only winter In eel larvae, ISH and RT-PCR assay showed that flounder amylase was enrolled in the GenBank trypsinogen and amylase mRNA expression starts database. The eel amylase precursor consisted of at 6 d.p.h. and that the expression level increased 512 amino acids, including a 15-amino-acid signal between 7 and 8 d.p.h.. The developmental pattern peptide, and its size and structure coincided with of trypsinogen was coincident with the result of that of winter flounder amylase. immunohistochemical analysis using anti-eel Pancreatic lipase is a major digestive enzyme trypsinogen.25 In contrast, mRNA expression of for lipids and has a structural similarity to hepatic lipase started at 8 d.p.h., 2 days later than trypsino- and lipoprotein lipase.17 All types of lipase have gen and amylase. Although RT-PCR is not suitable a common sequence, Gly-X-Ser-X-Gly,18 and this to analyze the exact quantitative differences in common sequence was also conserved in the eel mRNA expression, the lipase expression level was pancreatic lipase. clearly lower than that of trypsinogen and amylase In fish larvae, zymogen granules commonly even in the 10 d.p.h. larvae. Thus, it is supposed appear in the pancreatic cells by the first feeding; that the lipid digestive ability of early eel larvae for example, red sea bream Pagrus major,19 turbot is possibly poor. Because the lipid content of Pancreatic enzyme genes of eel FISHERIES SCIENCE 743

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