Kuroda’s sea hare Aplysia kurodai and laid egg were captured from the tide pools of Shimoda Bay (Shizuoka, Japan) in June.

The sequences of all primers are presented in Table 1. Total RNA was extracted from the ovotestis of A. kurodai using ISOGEN II reagent (Nippon Gene, Tokyo) according to the manufacturer’s instructions. First-strand cDNA was synthesized using 500 ng of total RNA and oligo dT primers with Prime Script Reverse Transcriptase (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Six degenerate primers were designed for the reverse transcriptase-polymerase chain reaction (RT-PCR) from conserved sequences of molluscan chitinase, including those from California sea hare (Aplysia californica; GenBank: XM_005112601), triangle sail mussel (Hyriopsis cumingii; GenBank: JN582038), Pacific oyster (Crassostrea gigas; GenBank: AJ971239), Hawaiian bobtail squid (Euprymna scolopes; GenBank: KF015222), and golden cuttlefish (Sepia esculenta; GenBank: AB986212).

The first PCR was performed using A. kurodai cDNA as a template and P1 and P2 as primers (Figure 1). The PCR parameters were as follows: 94˚C for 2 min, followed by 30 cycles of 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s. Nested PCR was performed using the products of the first PCR as templates and P3, P4, P5, and P6 as primers, with the same PCR parameters as described above. The nucleotide sequence analysis of the RT-PCR amplified chitinase cDNA fragments from the ovotestis of A. kurodai detected one nucleotide sequence (AkChi).

For the 3’ rapid amplification of cDNA ends (RACE), we designed primers specific to AkChi (i.e., P7, P8, and P9, respectively; Table 1) based on the detected sequences. We amplified cDNA fragments encoding the 3’ region of AkChi using A. kurodai cDNA as the template and the primer pairs P7 and 3R, P8 and 3R, and P9 and 3R (Figure 1). The PCR parameters were as follows: 94˚C for 2 min, followed by 30 cycles of 94˚C for 30 s, 56˚C for 30 s, and 72˚C for 30 s. For 5’ RACE, specific primers (P10, P11, and P12 for AkChi; Table 1) were designed based on the nucleotide sequences obtained from RT-PCR. cDNA fragments encoding the 5’ regions of AkChi were amplified using PCR. The first PCR was performed using the newly synthesized first-strand cDNA as a template and the primer pairs P10 and P11 for AkChi. Nested PCR was performed using the first PCR products as templates and the primer pairs P10 and P12 for AkChi. The PCR parameters were as follows:

Note: ^*Degenerate primers.

94˚C for 1 min, followed by 30 cycles of 94˚C for 30 s, 49˚C for 30 s, and 72˚C for 30 s.

The nucleotide sequences of cDNA fragments containing a full-length open reading frame (ORF) were confirmed by PCR using specific primers (P10 and P13 for AkChi; Table 1) and Platinum Pfx DNA Polymerase (Invitrogen, Carlsbad, CA).

The RT-PCR, 3’ RACE, and 5’ RACE amplification products, and the full-length amplification products were subcloned into pGEM-T Easy Vector (Promega, Madison, WI), according to the manufacturer’s instructions. Sequences were determined on an ABI PRISM 3130 genetic analyzer (Applied Biosystems, Foster City, CA) using a Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems).

A chitinase from the ovotestis of A. kurodai was purified as described [16] . The purified chitinase was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with AE-1360 EzStain Silver (ATTO, Tokyo). A gel slice was cut into small pieces and destained by destaining solution (15 mM K₃[Fe(CN)₆], 50 mM Na₂S₂O₃). Destained gel pieces were trypsinized as described in the manual of In-Gel Tryptic Digestion Kit manual (Thermo Scientific, Waltham, MA). The peptide mixtures thus obtained were subjected to a nano-scale liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC- ESI-MS/MS) analysis using a Q Exactive mass spectrometer (Thermo Scientific) equipped with a captive spray ionization source (Michrom Bioresources, Auburn, CA) and an Advance UHPLC System (Michrom Bioresources).

Total RNA was prepared from the ovotestis, egg, skin, gill, crop, anterior gizzard, and posterior gizzard as described in the cloning methods section (2.2) above. First-strand cDNA was pre-cloned from the RNA isolated from each tissue and egg as described in the RT-PCR section (2.2) above. For tissue-specific expression, we designed primers specific to AkChi (P14 and P15, respectively; Table 1) based on the detected sequences. AkChi was amplified using the first-strand cDNA as template and the primer pairs P14 and P15 (Table 1). The PCR parameters were as follows: 94˚C for 1 min, followed by 35 cycles of 94˚C for 30 s, 62˚C for 30 s, and 72˚C for 30 s. To determine the amount of total RNA in each tissue, we amplified β-actin mRNA fragments using specific primer pairs (Table 1).

In order to classify the chitinase from the ovotestis of A. kurodai among the GH family 18 chitinases, we constructed a phylogenetic tree based on the enzyme precursor sequences by the neighbor-joining method, using the ClustalW program (http://www.genome.jp/tools/clustalw/). A bacterial chitinase (GenBank: X03657) was used as the out group.

The structure of AkChi and the location of primer sequences are schematically represented in Figure 1. The internal sequence of the cDNA of A. kurodai ovotestis chitinase was amplified by RT-PCR using degenerate primers (from P1 to P6, respectively; Table 1); an amplified product of approx. 400 bp was obtained. The product was sequenced, and 86% homology with the acidic mammalian chitinase of A. californica was confirmed (accession no. XM_005112601). Because the sequence was part of ovotestis chitinase cDNA from A. kurodai, we used it to design gene-specific primers for 3’ and 5’ RACE (from P7 to P12; Table 1). An amplified product of approx. 430 bp was obtained by 3’ RACE, and its sequence contained a stop codon. An amplified product of approx. 520 bp was also obtained by 5’ RACE; its sequence contained a start codon. Based on these results, we designed full-length primers (P10 and P13; Table 1) to incorporate these start and stop codons. cDNA was amplified using the primers and the amplified product was sequenced.

The full-length cDNA of A. kurodai ovotestis chitinase (AkChi) was 1352 bp in length and contained an ORF of 1263 bp encoding 421 amino acids (Figure 2). The size of ORF of AkChi was smaller than it from H. cumingii [14] , 1962 bp encoding 653 amino acids. A poly-A sequence in eukaryotes was detected at the 3’ end of AkChi. AkChi, which encodes A. kurodai ovotestis chitinase, has been registered in the database of the DNA Data Bank of Japan (DDBJ) (accession no. LC085435). We compared the deduced amino acid sequence of AkChi with that of other organisms using BLAST, and the highest homology, 83%, was confirmed with the acidic mammalian chitinase of A. californica (accession no. XM_005112601). Figure 3 compares amino acid

sequences from AkChi and some other known molluscan chitinases (A. californica, H. cumingii, C. gigas, E. scolopes, and S. esculenta). The deduced amino acid sequence of AkChi was shown to have a structure of the GH family 18 chitinase, with an N-terminal signal peptide and a GH 18 catalytic domain. The catalytic domain also contained an active site that is a conserved sequence of GH family 18 chitinases (Figure 3). Though the chitinase of H. cumingii [14] and E. scolopes [15] had two chitin binding domains (CBDs) and the chitinase of S. esculenta had one CBD, AkChi lacked a CBD. It was reported that fish chitinases have one CBD [8] . This result suggests that the structure of molluscan chitinase is diverse compared to the fish chitinases.

We analyzed the sequences of the peptide fragments obtained by the tryptic treatment of the purified chitinase from the ovotestis of A. kurodai [16] were analyzed and compared them to the deduced amino acid sequence of AkChi. The obtained sequences from peptide fragments were consistent with the deduced amino acid sequence of AkChi (coverage: 35.39%, 119 residues) (Figure 2). This result suggests that AkChi is a gene coding the purified enzyme. In addition, trypsin is cut the C-terminal side of lysine and arginine. In this result, it was confirmed that the trypsin is working properly in the all of cleavage site.

We investigated the tissue-specific expression of AkChi in A. kurodai by RT-PCR using the housekeeping β-ac- tin gene as a control (Figure 4). It is reported that fish express chitinase to the digestive organs for digestion of chitin from food [17] . The expression profile results indicated that AkChi was present only in the ovotestis. We previously detected chitinase activity in the ovotestis and egg from A. kurodai [16] , whereas lysozyme activity (antibacterial enzyme activity) was not detected in any of the organs [16] . A. kurodai has to prey on seaweed.

Thus, A. kurodai is not necessary chitinase in digestion and attack of food as squid [10] [11] and octopus [12] , respectively. In addition, there is not necessary to shell formation because it does not even have shells. These results suggest that the role of this chitinase is as a defense against nematodes and fungus which have chitin in the body wall as a structural component.

We performed a phylogenetic tree analysis of GH family 18 chitinases and AkChi (Figure 5). Acidic mamma-

lian chitinases (AMCases) have been found in the stomach of mammals. Two chitinase groups with different structures and activity toward short substrates, AFCase-1 and AFCase-2, have been found in the stomach of fish [8] . Crustacean showed a chitinase group [18] . In contrast, molluscan chitinases did not show clear chitinase groups. The reason for this might be the differences in the chitinase domain structure that are due to the presence or absence of a CBD and the number of CBDs. We previously detected chitinase activity in the ovotestis and oviduct from the Walking sea hare Aplysia juliana [16] . If the success in cloning the chitinase from A. juliana, it will be conceivable to form a group of sea hare chitinase.