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Expression and activity of critical digestive enzymes during early larval development of the veined rapa whelk, Rapana venosa (Valenciennes, 1846)
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Expression and activity of critical digestive enzymes during early larval development of the veined rapa whelk, Rapana venosa (Valenciennes, 1846) Mei-Jie Yanga,b,c,d,1, Hao Songa,b,d,1, Zheng-Lin Yua,b,d, Yu-Cen Baie, Zhi Hua,b,c,d, Nan Huf, Cong Zhoua,b,c,d, Xiao-Long Wanga, Hai-Zhou Lig, Tao Zhanga,b,d,∗ a
CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China Laboratory for Marine Science and Technology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China c University of Chinese Academy of Sciences, Beijing, 100049, China d Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China e China Rural Technology Development Center, 54 Sanlihe Road, Beijing, 100045, China f Aquatic Ecology, Department of Biology, Lund University, Lund, Sweden g Shandong Fu Han Ocean Sci-Tech Co., Ltd, Haiyang, 265100, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rapana venosa Metamorphosis Cellulase Trypsin Food habit transition
Metamorphosis is a vital developmental event in the life cycle of molluscs and involves extensive morphological and physiological changes. Remodeling of the digestive system is suggested to occur anticipatorily to enable the larva to shift its diet (from filter feeding on microalgae to feeding on small bivalves) after metamorphosis. Changes in the profiles and activities of digestive enzymes, the main executors of digestion, can reflect substantial remodeling of the digestive system. Artificial aquaculture of Rapana venosa, an important commercial shellfish in China, has been hampered because the transition of its food habit during metamorphosis makes determining the timing and dose for bait regulation difficult. In the present study, full-length cDNA sequences encoding cellulase and trypsin were characterized, and cellulase and trypsin mRNA expression levels were analyzed. Additionally, patterns in the activities of six digestive enzymes, including trypsin and cellulase, were investigated throughout the early developmental stage of R. venosa. In the present study, the full-length cDNA of the cellulase gene, comprising 2,086 bp, was found to contain a 1,719-bp open reading frame encoding 572 amino acids, and the full-length cDNA of the trypsin gene was found to be 1,587 bp in length and contained an 855-bp open reading frame encoding 284 amino acids. Quantitative real-time PCR showed that the cellulase levels in R. venosa increased beginning at the early intramembrane veliger stage, whereas cellulase activity was significantly increased in the one-spiral whorl stage. The mRNA expression and activity of trypsin were greatly increased in the juvenile stage (postlarva), whereas those of cellulase were decreased during this stage, which indicated functional changes in the digestive system during larval food habit transition. Our results showed that remodeling of the digestive system occurs prior to metamorphosis and suggest that animal bait should be provided as early as possible to R. venosa in the four-spiral whorl stage to meet its nutritional requirements for the development of its digestive system and to ensure successful metamorphosis of competent larvae.
1. Introduction
commercial gastropod production in China (Ministry of Agriculture China, 2015). Researchers have found that hemocyanin from R. venosa possesses antiviral and antimicrobial activities and can be used against Epstein-Barr virus and as an alternative antimicrobial agent (Dolashka et al., 2011; Nesterova et al., 2011; Olga et al., 2015; Voelter et al., 2016). Great consideration has been given to the high economic and medicinal value of R. venosa. Unfortunately, R. venosa is highly threatened, and its population has greatly declined in recent years due to
The veined rapa whelk (Rapana venosa), a species native to temperate Asian waters ranging from the Sea of Japan, the Yellow Sea, the Bohai Sea and the East China Sea to Taiwan in the south (Mann and Harding, 2003), is an economically important fishery resource in China (Yuan, 1992). The annual production of R. venosa was approximately 0.1 million tons in 2014 and accounted for more than 20% of the
∗
Corresponding author. 7 Nanhai Road, Qingdao, Shandong, 266071, China. E-mail address: [email protected] (T. Zhang). 1 Contributes equally to this article. https://doi.org/10.1016/j.aquaculture.2019.734722 Received 29 May 2019; Received in revised form 2 October 2019; Accepted 8 November 2019 Available online 08 November 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Mei-Jie Yang, et al., Aquaculture, https://doi.org/10.1016/j.aquaculture.2019.734722
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stage, molluscs must break down plant-derived carbohydrates that are difficult to degrade; accordingly, cellulase is widely distributed in molluscs (Yokoe and Yasumasu, 1964; Xu et al., 2000; Nikapitiya et al., 2010; Li et al., 2009). Cellulase (endo-β-1,4-D-glucanase), a digestive enzyme that specifically hydrolyzes the internal β-1,4-glycoside linkages of cellulose chains (Tomme et al., 1995), is crucial for mollusc development (Suzuki et al., 2010; Niiyama and Toyohara, 2011). Previous studies have found that cellulase activity is greatly increased during the early development of molluscs, such as Babylonia areolata (Wei et al., 2007) and abalone (Jiang et al., 2012). Furthermore, the activities of proteases such as trypsin, which is a key alkaline protease secreted by the pancreas, are significantly changed during food habit transitions; these changes can be used as effective indicators of larval protein digestion. These changes in digestive mechanisms increase an organism's ability to withstand great variations in diet. Understanding these changes can help to determine the appropriate diet and timing of food habit transition. Several studies have examined changes in the digestive system of R. venosa during metamorphosis from the perspectives of transcriptomics and proteomics (Song et al., 2016a,b); however, no report on the ontogeny of digestive enzymes of R. venosa is available. The objectives of the present study were to (1) determine the mRNA expression and activity levels of major digestive enzymes in R. venosa during early development, (2) study the patterns in the activities of digestive enzymes to determine the features of food habit transition related to the metamorphosis of R. venosa and (3) identify digestive enzymes that can serve as indicators of R. venosa food habit transition. In the present work, the cDNA sequences of the cellulase and trypsin genes were analyzed to determine their temporal mRNA expression profiles, and the biochemical activities of six digestive enzymes essential for the digestive system and food habit transition during early development were detected. The present study provides insights into cellulase and trypsin dynamics during the early stages of life and could be helpful for designing diets and developing feeding strategies necessary to increase the larval survival of this species. Our study may also provide an improved understanding of the invasion mechanisms of carnivorous gastropods from the perspective of the digestive system, since invasion ability may be closely related to ingestion and digestion.
overexploitation and threats to its breeding grounds (Yang et al., 2007; Wei et al., 1999). Since 1992, efforts have been made to establish commercial aquaculture of the species due to its economic importance (Yuan, 1992). However, large-scale aquaculture of R. venosa has been seriously restricted by difficulties in rearing larvae to the juvenile stage, due to the mass mortality of planktonic larvae, which has become a major bottleneck in R. venosa aquaculture (Yuan, 1992; Yang et al., 2007). Therefore, it is necessary to understand the larval development process and establish technical procedures for early R. venosa larval culture to avoid overexploitation. As R. venosa is a mollusc, metamorphosis is a vital developmental event in its life cycle and occurs over a short time period (generally < 48 h). Metamorphosis is associated with high mortality (Huan et al., 2015), and the early larval phase is the most vulnerable stage in the R. venosa life cycle (Harding, 2006). R. venosa population dynamics are controlled by pre-metamorphosis recruitment and post-metamorphosis survival. Competent larvae need to find a suitable attachment site for metamorphosis, and an inappropriate substrate may be fatal (Bishop et al., 2006). Extensive morphological and physiological changes, such as velum degradation and reabsorption, foot proliferation and elongation, initiation of rapid secondary shell growth, and development of the radula, occur during metamorphosis. R. venosa larvae exhibit several characteristics that differ from those of other molluscs; these include a longer metamorphosis stage (30–35 days) and a larger size (1400–1500 μm) than those of the competent larvae of most other molluscs (Wei et al., 1999; Pan et al., 2013) and transition of food habits from herbivory to carnivory. This transition does not occur in lifelong herbivorous gastropods and is crucial for the metamorphosis of R. venosa larvae. In our previous study, we found that the mass mortality of larvae and juveniles mainly resulted from the lack of or delay in appropriate feeding, which can result in cannibalism and failure of metamorphosis (Yu et al., 2018). Therefore, appropriate feeding at the appropriate time can efficiently prevent mortality caused by cannibalism and metamorphosis failure. Thus, to establish technical procedures for early R. venosa larval culture, research on the development of the digestive system is required. Successful digestive system development is significant for the survival and growth of larvae because an efficient digestive system enables the larvae to capture, ingest, digest and absorb food (Kjorsvik et al., 2004). Thus, morphological and functional changes in the digestive system play vital roles in the metamorphic transition of R. venosa from a diet of microalgae to one of bivalve molluscs (Pan et al., 2013). Previous studies have found that many genes and proteins associated with food intake and digestion, including some digestive enzymes, are differentially expressed between the larval and postlarval stages in R. venosa (Song et al., 2016 a), and researchers have shown that phytophagous digestive enzymes (such as cellulase) are downregulated after metamorphosis, whereas carnivorous digestive enzymes (such as trypsin) are upregulated. Ontogenetic changes in the types and concentrations of digestive enzymes, which are indicative of shifts in the ability to digest dietary components, have been used to identify dietary shifts in a range of invertebrates (Hammer and Watts, 2000; Johnston, 2003). For example, changes in digestive enzyme activity and hepatopancreatic development in Macrobrachium rosenbergii occur when individuals shift from carnivorous to omnivorous habits during the larval period (Kamarudin et al., 1994). Similarly, changes in protease and αamylase activities occur in the black tiger prawn Penaeus monodon during the transitions from herbivory in the nauplius stage to carnivory in the mysis stage and then to omnivory in juveniles and adults, reflecting changes in feeding preference. Although changes in feeding preference are common in invertebrates, few studies have been conducted on the ontogenetic changes in digestive enzymes that occur in R. venosa; such studies may assist the identification of critical ages and sizes related to dietary changes in this species (Song et al., 2016 b). Most molluscs need to undergo a short or lifelong herbivorous stage, during which they feed mainly on microalgae or other plants. In this
2. Materials and methods 2.1. Larval rearing and sample preparation Larvae of R. venosa were obtained from Blue Ocean Co., Limited (Laizhou, Shandong, China). The parent culture, spawn, intramembrane larvae and planktonic larvae were cultured at a density of 0.1 ind/ml in 3 × 5 × 1.5 m cement pools at 24.6–25.6 °C according to the methods of Yang et al. (2007). Isochrysis galbana, Chlorella vulgaris and Platymonas subcordiformis were mixed and served as the diet for the planktonic larvae. The average shell height (or mean size of the intramembrane larva) and the daily increase in shell height were measured in 30 randomly selected larvae every day following the methods of Pan et al. (2013). Intramembrane larval samples were collected from 1 to 13 days after spawning (das) at seven major developmental stages: the cleavage stage (c), the blastula stage (b), the gastrula stage (g), the trochophore stage (t), the early intramembrane veliger stage (ev), the middle intramembrane veliger stage (mv), and the later intramembrane veliger stage (lv). Planktonic larval and postlarval samples were collected from 14 to 60 das at five major developmental stages: the one-spiral whorl (V–I) stage, the two-spiral whorl (VII) stage, the three-spiral whorl (V-III) stage, the four-spiral whorl (competent larva, V-IV) stage, and the juvenile (postlarva, J) stage. The samples were monitored under a microscope to ensure developmental synchrony. Whole larval samples at each development stage were taken in triplicate and octuplicate for molecular and biochemical analyses, respectively, since the individual larvae were too small and difficult to 2
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from the GenBank database, and DNAMAN software was then used to perform multiple-sequence alignments. Phylogenetic trees were constructed by MEGA7.0 using the neighbor-joining (N-J) method.
dissect, and each sample was then washed with PBS, snap-frozen in liquid nitrogen, and stored at −80 °C until use. 2.2. Extraction of total RNA
2.5. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from all the larval samples using a TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan) to clone fulllength cDNA and analyze the mRNA expression of the cellulase and trypsin genes following the manufacturer's protocol. The purity and concentration of the RNAs were measured by a Nanodrop spectrophotometer (Thermo Scientific, USA) and gel electrophoresis.
Primers for qRT-PCR were designed based on the full-length cDNA sequences of the cellulase and trypsin genes; RT-Cel-F and RT-Cel-R were used as forward and reverse primers, respectively, for the cellulase gene, and RT-Try-F and RT-Try-R were used as forward and reverse primers, respectively, for the trypsin gene. The 60S ribosomal protein L28 (RL28-F, RL28-R) was used as a housekeeping gene for signal normalization within the experiment (Song et al., 2017). First-strand cDNAs for qRT-PCR were synthesized using reverse transcriptase (TaKaRa). A SYBR Green-based real-time PCR assay (SYBR PrimeScript™ RT-PCR Kit II, TaKaRa) was used with an Eppendorf Mastercycler® ep Realplex (Eppendorf, Hamburg, Germany) for qRT-PCR analysis. Reactions containing 10 μl of Maxima™ SYBR Green qPCR master mix (TaKaRa), 10 pmol of forward or reverse primer in a volume of 0.5 μl each, 1 μl (100 ng) of cDNA and 8 μl of sterile deionized water were prepared in a total volume of 20 μl. Amplifications were performed according to the manufacturer's instructions. Standard curves were made with cDNA template diluted 10, 102, 103, 104, and 105 fold. qRTPCR was performed using the following thermocycler program: 95 °C for 5 s and 40 cycles of 95 °C for 15 s and 60 °C (cellulase) or 58 °C (trypsin) for 30 s. Relative gene expression was calculated using the 2−ΔΔCt method.
2.3. Cloning of the full-length cDNAs for the cellulase and trypsin genes 2.3.1. Cloning of the middle fragments of the full-length cDNAs Primer pairs for the middle fragments of the cellulase and trypsin genes based on expressed sequence tags in the transcriptome library of R. venosa (Song et al., 2016a,b) were designed using Primer Premier 5.0 software and synthesized by Tsingke (China). All of the primers used in this study are listed in Supplementary Table 1. First-strand cDNA was synthesized using a Prime Script™ RT Reagent Kit with gDNA Eraser (TaKaRa, Japana), and the RNA used for first-strand cDNA synthesis was a mixture of RNA samples from R. venosa specimens at all developmental stages. The middle fragments of the two genes were amplified by polymerase chain reaction (PCR) using Ex Taq DNA polymerase (TaKaRa). The PCR program implemented in a thermal cycler (TaKaRa) was as follows: initial denaturation at 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 55 °C (cellulase) or 57 °C (trypsin) for 30 s and 72 °C for 40 s and a final extension at 72 °C for 10 min. PCR products were purified using a 1% agarose gel with a 2,000 bp ladder used as a marker. Bands of the expected sizes were excised from the gel and subsequently cloned into the pMD19-T vector (TaKaRa) and sequenced by Tsingke (China), and their sequences were used to query GenBank.
2.6. Digestive enzyme activity assays Whole larval samples were collected and homogenized for biochemical analysis of the enzymes. After homogenization, each sample was centrifuged at 4 °C and 3000×g for 10 min, and the supernatant was collected and stored at −20 °C. The soluble protein content of all the samples was determined by the method using bovine serum albumin as a standard. The cellulase, trypsin, pepsin, chymotrypsin, αamylase and lipase activities were evaluated using corresponding assay kits (Nanjing Jiancheng, Bioengineering Institute, China), and the results of the enzyme assays were expressed as specific activity in units per mg protein (U/mg protein). Cellulase activity was measured using a cellulase assay kit (A138) with the 3,5-dinitrosalicylic acid (DNS) method. The chromogenic reaction was incubated in boiling water for 15 min, and the absorbance of the samples at a wavelength of 550 nm was measured. One unit of cellulase was defined as 1 mg of enzyme, which hydrolyzed the substrate and generated 1.0 μg of glucose in 1 min. Trypsin activity was detected by a trypsin assay kit (A080-2) based on ultraviolet colorimetry. Arginine ethyl was used as the substrate, and the reaction was performed at 37 °C. The absorbance of the samples at a wavelength of 253 nm was read at 30 s and 20 min 30 s to analyze trypsin activity. One unit of trypsin was defined as 1 mg of enzyme, which generated a change in optical density (OD) of 0.003 in 1 min at 37 °C. A chymotrypsin assay kit (A080-3) was used to analyze chymotrypsin activity using casein as the substrate by reading the absorbance of the samples at a wavelength of 660 nm, and 1 U of chymotrypsin was defined as 1 mg enzyme, which produced 1 μg amino acid in 1 min at 37 °C, pH 8. An α-amylase assay kit (A016-1) was used to analyze α-amylase activity based on starch-iodine colorimetry by reading the absorbance of the samples at a wavelength of 660 nm. One unit of α-amylase was defined as 1 mg of enzyme, which hydrolyzed 10.0 mg of starch in 30 min. Lipase activity was determined using a lipase assay kit (A054-2) with methyl-halogen as the substrate by continuously monitoring the absorbance of the samples at a wavelength of 570 nm for 3 min at 37 °C following the manufacturer's instructions. The rates of change in the absorbance were recorded to analyze lipase activity. Pepsin activity was determined using a pepsin assay kit (A0801) with casein as a substrate following the manufacturer's instructions.
2.3.2. Cloning of the 5′ and 3’ fragments of the full-length cDNAs First-strand cDNA (5′- and 3′-rapid amplification of cDNA ends (RACE)-ready cDNAs) synthesis and RACE were performed using a SMARTer™ RACE cDNA Amplification Kit (Clontech, USA). The primers used for RACE were designed based on the middle fragment sequences determined above using Primer Premier 5 software. The 5′ ends of the cellulase and trypsin genes were amplified with the primers try-5′GSPn and cel-5′- GSPn, respectively, and a universal primer mix (UPM), and the 5′-RACE-ready cDNAs were used as the templates. The 3′ ends of the cellulase and trypsin genes were amplified using the primers try3′- GSPn and cel-3′- GSPn, respectively, and a UPM, and the 3′RACEready cDNAs were used as the templates. PCR amplification to confirm the full-length cDNAs was conducted as follows: 94 °C for 2 min; 35 cycles of 94 °C for 30 s, 50–60 °C for 30 s, and 72 °C for 1 min; and 72 °C for 10 min (Eppendorf). PCR products were inserted into the pMD19-T vector (TaKaRa) and used to transform JM109 competent cells (TaKaRa) according to the manufacturer's instructions, and the nucleotide sequences of 10 positive clones were confirmed by Sangon Biotech (China). The sequences were analyzed and assembled by DNAStar software to obtain the full-length cDNA sequences. 2.4. Sequence and phylogenetic analyses The full-length cDNAs and protein sequences of the cellulase and trypsin genes were analyzed using the corresponding National Center for Biotechnology Information BLAST programs (http://www.ncbi.nlm. nih.gov/BLAST/). Functional sites or domains in the protein sequences were predicted using the SMART program (http://smart.emblheidelberg.de/). The molecular masses and theoretical isoelectric points of the putative proteins were predicted using ExPasy Compute pI/Mw software (http://www.expasy.org/tools/pi_tool.html). The cDNA sequences of the two genes from different species were obtained 3
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Fig. 1. Larval growth in terms of shell height (a) and daily increase in shell height (b) during R. venosa larval development for up to 60 das. Data are expressed as the mean ± SE.
The chromogenic reactions were performed at 37 °C for 20 min, and the absorbance of the samples at a wavelength of 660 nm was read to analyze pepsin activity. One unit of pepsin was defined as 1 mg of enzyme, which produced 1 μg of amino acid in 1 min at 37 °C and pH 3.
intramembrane larvae to planktonic larvae was significantly lower than the growth rate before this transition. In contrast, the growth rate was very high in planktonic larvae, especially in those in the V-II and V-IV stages.
2.7. Data analysis
3.2. Identification and analysis of the cellulase and trypsin genes
The larval growth data, qRT-PCR expression results and the biochemical enzyme activities of cellulase, trypsin, chymotrypsin, α-amylase, lipase and pepsin were analyzed by one-way ANOVA with significance indicated by P < 0.05. Tukey's test was performed to determine normality, and equality of variances was assessed by Levene's test before ANOVA was performed. All statistical analyses were preformed using SPSS 19.0 software (SPSS Inc., USA).
The full-length cDNA of the cellulase gene is 2,086 bp in length, comprising a 122-bp 5′ UTR, a 245-bp 3′ UTR, and a 1,719-bp ORF encoding 572 amino acids (GenBank accession no. MK850389) (Fig. 2 a). The calculated molecular mass and predicted isoelectric point of the cellulase protein are 63.76 kDa and 6.62, respectively. The predicted protein sequence of cellulase, a typical modularized enzyme, contains an N-terminal CBM4_9_ carbohydrate-binding domain and a C-terminal glycoside hydrolase GHF10 catalytic domain (GHF10). The full-length cDNA of the trypsin gene is 1,587 bp in length, comprising a 93-bp 5′ UTR, a 639-bp 3’ UTR, and an 855-bp ORF encoding 284 amino acids (GenBank accession no. MK850390) (Fig. 3 a). The calculated molecular mass and predicted isoelectric point of the trypsin protein are 30.58 kDa and 8.78, respectively. Domain architecture analysis indicated that the trypsin gene contains a signal peptide (amino acids 1–46) and the amino acid sequence of the trypsin-like serine protease (Tryp_SPc) family domain (amino acids 47–279).
3. Results 3.1. Larval growth The growth of R. venosa larvae was measured at 0–60 das by determining the shell height (Fig. 1 a) and calculating the daily increase in shell height (Fig. 1 b). The daily growth rate of the intramembrane larvae was low, and the growth rate during the transition from 4
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Fig. 2. Characteristics of the amino acid sequence deduced from the cellulase gene. (a) The signal peptide sequence (1–20) and the amino acid sequences of the CBM4_9_ (21–150) and GHF10 (258–507) domains are denoted by red, blue and green boxes, respectively, and the active site amino acids are denoted by green triangles. (b) Multiple alignment of amino acid sequences deduced by cellulase genes. Pomacea canaliculate [PVD26373.1], Branchiostoma belcheri [XP_019626719.1], Paenibacillus ihumii [WP_055105560.1], Triticum urartu [EMS67873.1], Brachypodium distachyon [XP_003560192.1], Lingula anatina [XP_013410933.2]. (c) Phylogenetic tree based on the amino acid sequences of cellulase proteins from R. venosa and other species. The tree was constructed based on multiple sequences generated by Clustal X and aligned using the N-J method in MEGA 7.0. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3. Homology and phylogenetic analyses of the cellulase and trypsin genes
used for homology and phylogenetic analyses. A BLAST search showed that the predicted amino acid sequence of cellulase from R. venosa shared the highest sequence identity with cellulase from Pomacea
Cellulase and trypsin protein sequences from different species were 5
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Fig. 2. (continued)
(WP_055105560.1). Compared with the sequence of cellulase, the sequence of trypsin from R. venosa shared low similarity with the trypsin sequences of other species. The sequence identity of trypsin from R. venosa with trypsin sequences from P. canaliculata (PVD20786.1),
canaliculata (XP_025105693.1, 59.64% identity), followed by cellulases from Branchiostoma belcheri (XP_019626719.1, 41.56% identity), Lingula anatine (XP_013410933.2, 35.85% identity), Brachypodium distachyon (XP_019627090.1, 33.58) and Paenibacillus ihumii 6
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Fig. 3. Characteristics of the amino acid sequence deduced from the trypsin gene. (a) The signal peptide (amino acids 1–46) and the amino acid sequence of the Tryp_SPc domain (amino acids 46–283) are denoted by red and blue boxes, respectively, and the signal peptide breaking site, substrate binding site and active site are denoted by green, blue and red triangles, respectively. (b) Multiple alignment of amino acid sequences deduced from trypsin genes. Pomacea_canaliculata [PVD36883.1], Biomphalaria_glabrata [XP_013088769.1], Drosophila_bipectinata [XP_017105525.1], Pelodiscus_sinensis [XP_006120382.1], Zonotrichia_albicollis [XP_026654917.1], Bos_taurus [DAA30409.1], Mus_ musculus [NP_001019869.1]. (c) Phylogenetic tree based on trypsin amino acid sequences from R. venosa and other species. The tree was constructed based on multiple sequences generated by Clustal X and aligned using the N-J method in MEGA 7.0. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
cellulase and trypsin genes in R. venosa at different developmental stages. The cellulase gene showed very low expression during the early developmental stages, and a significant increase in cellulase gene expression occurred from the early larval stage to the planktonic larval stage (P < 0.05); a high level of cellulase gene expression was maintained throughout the latter stage (Fig. 4 a). However, a significant decrease in cellulase gene expression occurred at the postlarval stage. Trypsin mRNA levels were detectable during the early stages of development; trypsin expression was high in the c stage and then gradually decreased with larval growth. Trypsin mRNA was expressed at low levels in the whole planktonic larval stage, whereas significantly (P < 0.05) high trypsin mRNA expression levels, almost identical to those at the c stage, were observed in the postlarval stage (Fig. 4 b).
Pocillopora damicornis (XP_027047043.1), Parasteatoda tepidariorum (XP_021001494.1), Xiphophorus maculatus (XP_023205443.1), Penaeus vannamei (ROT79329.1) and Trichuris suis (KHJ42080.1) was 41.15%, 39.84%, 39.18%, 37.55%, 35.25% and 34.71%, respectively. The deduced cellulase and trypsin protein sequences were used for multiplesequence alignments using the ClustalW sequence alignment program (Figs. 2 b and Fig. 3 b). To further clarify the evolutionary relationships between the R. venosa cellulase and trypsin genes and those of other species, we selected > 20 species for phylogenetic analyses and constructed phylogenetic trees by the N-J method. In the phylogenetic tree for the cellulase genes, R. venosa cellulase clusters with the cellulase genes from P. canaliculata and Ampullaria crossean (Fig. 2 c). Trypsin from R. venosa clusters with the trypsin genes from P. canaliculata and Lottia gigantea, showing low similarity with trypsin genes from other species (Fig. 3 c). The cellulase and trypsin genes from R. venosa both belong to the Mollusca cluster and are closely related to those of other gastropods.
3.5. Digestive enzyme activities In the present study, enzyme activities were detectable at as early as the hatching stage. Low levels of cellulase activity were observed during the intramembrane larval stages, and the levels increased from the V–I stage, with a high level of cellulase activity maintained throughout the planktonic larval stage. Cellulase activity peaked at the
3.4. Digestive enzyme gene expression determined by qRT-PCR qRT-PCR was performed to detect the mRNA expression levels of the 7
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Fig. 3. (continued)
the V–I larval stage, with the level subsequently maintained throughout the planktonic larval stage (P < 0.05). A large increase in trypsin activity occurred in the postlarval stage, and trypsin activity showed peak levels throughout the larval stage (P < 0.05) (Fig. 5 b).
V-III stage and then decreased, with a significant decrease in cellulase activity observed in the postlarval stage (P < 0.05); cellulase activity was lowest in the whole larval stage (Fig. 5 a). Low trypsin activity was observed at early developmental stages and significantly increased in 8
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Fig. 4. qRT-PCR analysis of cellulase and trypsin mRNA expression during R. venosa larval development (mean ± SE, n = 3). Different superscripted letters indicate significant differences (P < 0.05).
(Icely and Nott, 1992; Ceccaldi, 1997; Jones et al., 1997a; 1997b). For instance, high protease activity indicates protein catabolism, and cellulase activity suggests carbohydrate (i.e., starch and glycogen) catabolism (Kamarudin et al., 1994; Rodriguez et al., 1994; Johnston, 2003). The timing and quantity of digestive enzyme secretion reflect the development of the digestive system. Additionally, the larval feeding pattern is directly related to the ontogeny of digestive enzymes. Digestive enzymes in aquatic animals, especially fish and crustaceans, have been widely researched, whereas relatively little research has been conducted on digestive enzymes in molluscs (Yang et al., 1998), and few studies have focused on changes in digestive enzymes during individual development. Previous studies have indicated that significant changes in digestive enzymes occur during development in abalone and B. areolata (Bansemer et al., 2016; Jiang et al., 2012; Wei et al., 2007). Therefore, digestive enzymes might be useful as indicators of the early
In addition to the cellulase and trypsin activities, the activities of chymotrypsin, α-amylase, lipase and pepsin were detected. The chymotrypsin and pepsin activities ranged from 2 to 10 U mg−1 protein, whereas the activity of α-amylase ranged from 0.2 to 1 U mg−1 protein and that of lipase ranged from 0.04 to 0.08 U mg−1 protein. Although the activity of α-amylase increased significantly before the planktonic larval stage and peaked at the lv stage, all four digestive enzymes were active at low levels throughout the early development stage.
4. Discussion Changes in digestive enzymes during the early development stage can provide useful information about individual development because digestive enzymes are responsible for the breakdown of nutrient reserves to provide energy for maintenance, development and growth 9
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Fig. 5. Specific (a) cellulase, (b) trypsin, (c) chymotrypsin, (d) α-amylase, (e) lipase and (f) pepsin activities during the development of larval R. venosa. (means ± SEs, n = 8). Different superscripted letters indicate significant differences (P < 0.05).
canaliculata (Imjongjirak et al., 2008). These differences in the GHF subfamily suggest that the cellulase proteins in gastropods might have separated from those in other molluscs during evolution. In contrast, the full-length cDNA sequence of trypsin from R. venosa showed low degrees of sequence homology with those from other animals (< 50%) and showed the highest degree of sequence similarity with a hypothetical protein from P. canaliculata (41.15%). The Tryp_SPc family is ubiquitous in animals and plays diverse roles, especially in the digestive system (Wu et al., 2009). However, there has been little research on trypsin in molluscs. The trypsin cloned from R. venosa may be a new type of trypsin-like serine protease in molluscs. As in most gastropods (Liu and Sun, 2008; Zhou et al., 1996; Zhou et al., 2000), in R. venosa, early development comprises three stages: an intramembrane larval stage, a planktonic larval stage and a postlarval stage. Digestive enzymes differ significantly depending on the developmental stage, especially at the critical points of different developmental stages. Throughout the intramembrane larval stage, R. venosa
development and metamorphosis of gastropods that undergo food habit transition. To further understand the development of the digestive system in the early development and metamorphosis of R. venosa, we investigated changes in cellulase and trypsin mRNA expression and bioactivity during R. venosa growth. In the present study, the full-length cDNA sequences of the cellulase and trypsin genes were cloned from R. venosa, and cDNA libraries were generated from samples containing R. venosa larvae at different developmental stages. Cellulase from R. venosa showed high degrees of sequence homology with cellulases from other molluscs, particularly P. canaliculata. Cellulase from R. venosa contains a catalytic domain that is a conserved 250-amino acid region that belongs to the GHF10 subfamily, whereas most reported cellulases from animals contain a catalytic domain belonging to the GHF9, GHF5 or GHF45 subfamily (Byrne et al., 1999; Yan et al., 1998; Xu et al., 2001). GHF10, a multifunctional cellulase generally involved in the microbial degradation of cellulose and xylans, was first isolated from the animals A. crossean and P. 10
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important interim period during which the expression of different kinds of digestive enzymes is regulated (Song et al., 2016 b), of which cellulase and trypsin can serve as major indicators. During food habit transition, larvae spend their energy on not only the further development of digestive organs but also velum degradation and reabsorption, foot proliferation and elongation, initiation of rapid secondary shell growth, development of the radula, and the search for suitable attachment sites. Thus, the larval growth rate decreases significantly during this transition. A previous study indicated that large-molecule protein feeding is required for the development of pancreatic digestive functions in rats (Kinouchi et al., 2012). Therefore, it may be very important to provide suitable and adequate animal bait as early as possible in the V-IV stage (competent larva) for the larvae to undergo changes in their digestive systems, metamorphosis, and further growth and development.
larval growth increased steadily and slowly, beginning at 1 das, which is consistent with the growth patterns of other gastropod larvae (Oyarzun and Strathmann, 2011). R. venosa larval growth was extremely slow during the transition from the intramembrane larval stage to the planktonic larval stage, and we speculate that the larvae need large amounts of energy to complete the hatching process. Correspondingly, both cellulase and trypsin activities were low during this stage because the larvae do not ingest any food and are wholly reliant on endogenous reserves, which is also the case for other gastropods (Zhao et al., 2012; Oyarzun and Strathmann, 2011). However, in the present study, the mRNA expression of cellulase was greatly increased beginning at the ev stage and peaked before hatching (P < 0.05), so we speculate that the digestive organs may begin to appear at this stage. Larval growth is dependent upon the successful development of the digestive system during the most critical first feeding phase (Mir et al., 2018), so development of the digestive system during the intramembrane larval stage is significant for growth during the early development stage. Molluscs are biphasic, and most of them need go through a planktonic larval period after hatching (Huan et al., 2015). During this period, the larvae mainly feed on microalgae, such as the diatoms I. galbana, C. vulgaris and P. subcordiformis, which contain plentiful cellulose. Cellulose is mainly degraded by cellulase endogenous to the mollusc digestive system. Therefore, the increase in cellulase mRNA expression before hatching may suggest that the initial development of the digestive system is complete and that the larvae are capable of digesting microalgae for planktonic life. Relative to the change in cellulase mRNA expression, the change in cellulase activity was delayed until the V–I stage. We speculate that this asynchrony may have been due to the stimulation of digestive enzyme activity by food. (Ou and Liu, 2007; Genodepa et al., 2018). Therefore, adequate and appropriate microalgal feed should be maintained in the hatching tanks of larvae to ensure abundant food upon hatching. In addition to being necessary for larval growth and development, abundant food can promote further development of the larval digestive system. Throughout the planktonic larval stage, the larval growth rate was significantly higher than the growth rate of the intramembrane larvae, whereas a decrease in the growth rate was observed at the postlarval J stage. Meanwhile, mRNA expression and cellulase activity were maintained at high levels, which reflected the microalgal diet. Additionally, a significant increase in trypsin activity was observed at the V–I stage because protein is necessary for animal development; microalgae contain large amounts of plant proteins in addition to carbohydrates. At the end of the gastropod planktonic larval stage, gastropod larvae need to undergo metamorphosis to enter the postlarval stage and develop into juveniles. A previous study indicated that the mRNA expression of cellulase and three other polysaccharide lyase genes in Haliotis discus hannai Ino was significantly increased from the early peristomial shell larval stage, which is consistent with the development of the digestive system and the appearance of new digestive organs during metamorphosis (He, 2011). However, a substantial decrease in cellulase expression was observed during B. areolata metamorphosis (Wei et al., 2007). The reason for this difference is that abalone is herbivorous, whereas B. areolata is carnivorous. In the present study, the mRNA expression of cellulase was slightly decreased in the V-IV stage (competent larvae), and the mRNA expression and activity of cellulase reached very low levels in the J stage (postlarva), consistent with a previous study on B areolata (Wei et al., 2007). In contrast, the mRNA expression and activity of trypsin remained low in the V-IV stage (competent larva) but greatly increased when the larvae underwent metamorphic transition. Relative to the cellulase and trypsin activities, the activities of the other four digestive enzymes in this process remained very low, with no pronounced changes in activity. This finding supports the conjecture in previous research that changes in the regulation of these digestive enzymes are responsible for the change in diet after metamorphosis and that the V-IV stage (competent larvae) is an
5. Conclusion Although the mRNA expression of cellulase increased beginning at the ev stage, a significant increase in cellulase activity was observed at the V–I stage. This significant increase suggests that the initial development of the digestive system is complete at this stage and that the larvae are capable of digesting microalgae for planktonic life. Additionally, the mRNA expression and activity of trypsin were greatly increased in the J stage (postlarva), whereas those of cellulase were decreased, which reflected the food habit transition from herbivory to carnivory. The activities of cellulase and trypsin were 10 to 104 times greater than those of the other four digestive enzymes studied. This finding suggests that cellulase and trypsin are the major digestive enzymes in R. venosa and can serve as indicators for the development of the digestive system and food habit transition during the early development of R. venosa. Based on our results, we suggest that animal bait be provided as early as possible in the V-IV stage to promote the development of R. venosa and ensure successful metamorphosis. Integrative studies on the histological organization of digestive structures and histochemical localization studies are needed to completely understand digestive development in R. venosa.
Declaration of competing interest The authors report no conflict of interest.
Acknowledgments The authors thank the Ma Shan Group Co., Ltd., for providing support with the experimental material. This research was supported by the National Natural Science Foundation of China (grant no. 31572636), the Natural Science Foundation of Shandong Province (grant no. ZR2019BD003), the China Postdoctoral Science Foundation (grant no. 2019M652498), the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-49), the Industry Leading Talents Project of Taishan Scholars (Recipient: Tao Zhang), the ‘Double Hundred’ Blue Industry Leader Team of Yantai (Recipient: Tao Zhang), and the Creative Team Project of the Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology (no. LMEES-CTSP-2018-1). The funders had no role in the study design, data collection or analysis, decision to publish or preparation of the manuscript.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734722. 11
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Expression and activity of critical digestive enzymes during early larval development of the veined rapa whelk, Rapana venosa (Valenciennes, 1846) Mei-Jie Yanga,b,c,d,1, Hao Songa,b,d,1, Zheng-Lin Yua,b,d, Yu-Cen Baie, Zhi Hua,b,c,d, Nan Huf, Cong Zhoua,b,c,d, Xiao-Long Wanga, Hai-Zhou Lig, Tao Zhanga,b,d,∗ a
CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China Laboratory for Marine Science and Technology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China c University of Chinese Academy of Sciences, Beijing, 100049, China d Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China e China Rural Technology Development Center, 54 Sanlihe Road, Beijing, 100045, China f Aquatic Ecology, Department of Biology, Lund University, Lund, Sweden g Shandong Fu Han Ocean Sci-Tech Co., Ltd, Haiyang, 265100, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rapana venosa Metamorphosis Cellulase Trypsin Food habit transition
Metamorphosis is a vital developmental event in the life cycle of molluscs and involves extensive morphological and physiological changes. Remodeling of the digestive system is suggested to occur anticipatorily to enable the larva to shift its diet (from filter feeding on microalgae to feeding on small bivalves) after metamorphosis. Changes in the profiles and activities of digestive enzymes, the main executors of digestion, can reflect substantial remodeling of the digestive system. Artificial aquaculture of Rapana venosa, an important commercial shellfish in China, has been hampered because the transition of its food habit during metamorphosis makes determining the timing and dose for bait regulation difficult. In the present study, full-length cDNA sequences encoding cellulase and trypsin were characterized, and cellulase and trypsin mRNA expression levels were analyzed. Additionally, patterns in the activities of six digestive enzymes, including trypsin and cellulase, were investigated throughout the early developmental stage of R. venosa. In the present study, the full-length cDNA of the cellulase gene, comprising 2,086 bp, was found to contain a 1,719-bp open reading frame encoding 572 amino acids, and the full-length cDNA of the trypsin gene was found to be 1,587 bp in length and contained an 855-bp open reading frame encoding 284 amino acids. Quantitative real-time PCR showed that the cellulase levels in R. venosa increased beginning at the early intramembrane veliger stage, whereas cellulase activity was significantly increased in the one-spiral whorl stage. The mRNA expression and activity of trypsin were greatly increased in the juvenile stage (postlarva), whereas those of cellulase were decreased during this stage, which indicated functional changes in the digestive system during larval food habit transition. Our results showed that remodeling of the digestive system occurs prior to metamorphosis and suggest that animal bait should be provided as early as possible to R. venosa in the four-spiral whorl stage to meet its nutritional requirements for the development of its digestive system and to ensure successful metamorphosis of competent larvae.
1. Introduction
commercial gastropod production in China (Ministry of Agriculture China, 2015). Researchers have found that hemocyanin from R. venosa possesses antiviral and antimicrobial activities and can be used against Epstein-Barr virus and as an alternative antimicrobial agent (Dolashka et al., 2011; Nesterova et al., 2011; Olga et al., 2015; Voelter et al., 2016). Great consideration has been given to the high economic and medicinal value of R. venosa. Unfortunately, R. venosa is highly threatened, and its population has greatly declined in recent years due to
The veined rapa whelk (Rapana venosa), a species native to temperate Asian waters ranging from the Sea of Japan, the Yellow Sea, the Bohai Sea and the East China Sea to Taiwan in the south (Mann and Harding, 2003), is an economically important fishery resource in China (Yuan, 1992). The annual production of R. venosa was approximately 0.1 million tons in 2014 and accounted for more than 20% of the
∗
Corresponding author. 7 Nanhai Road, Qingdao, Shandong, 266071, China. E-mail address: [email protected] (T. Zhang). 1 Contributes equally to this article. https://doi.org/10.1016/j.aquaculture.2019.734722 Received 29 May 2019; Received in revised form 2 October 2019; Accepted 8 November 2019 Available online 08 November 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Mei-Jie Yang, et al., Aquaculture, https://doi.org/10.1016/j.aquaculture.2019.734722
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stage, molluscs must break down plant-derived carbohydrates that are difficult to degrade; accordingly, cellulase is widely distributed in molluscs (Yokoe and Yasumasu, 1964; Xu et al., 2000; Nikapitiya et al., 2010; Li et al., 2009). Cellulase (endo-β-1,4-D-glucanase), a digestive enzyme that specifically hydrolyzes the internal β-1,4-glycoside linkages of cellulose chains (Tomme et al., 1995), is crucial for mollusc development (Suzuki et al., 2010; Niiyama and Toyohara, 2011). Previous studies have found that cellulase activity is greatly increased during the early development of molluscs, such as Babylonia areolata (Wei et al., 2007) and abalone (Jiang et al., 2012). Furthermore, the activities of proteases such as trypsin, which is a key alkaline protease secreted by the pancreas, are significantly changed during food habit transitions; these changes can be used as effective indicators of larval protein digestion. These changes in digestive mechanisms increase an organism's ability to withstand great variations in diet. Understanding these changes can help to determine the appropriate diet and timing of food habit transition. Several studies have examined changes in the digestive system of R. venosa during metamorphosis from the perspectives of transcriptomics and proteomics (Song et al., 2016a,b); however, no report on the ontogeny of digestive enzymes of R. venosa is available. The objectives of the present study were to (1) determine the mRNA expression and activity levels of major digestive enzymes in R. venosa during early development, (2) study the patterns in the activities of digestive enzymes to determine the features of food habit transition related to the metamorphosis of R. venosa and (3) identify digestive enzymes that can serve as indicators of R. venosa food habit transition. In the present work, the cDNA sequences of the cellulase and trypsin genes were analyzed to determine their temporal mRNA expression profiles, and the biochemical activities of six digestive enzymes essential for the digestive system and food habit transition during early development were detected. The present study provides insights into cellulase and trypsin dynamics during the early stages of life and could be helpful for designing diets and developing feeding strategies necessary to increase the larval survival of this species. Our study may also provide an improved understanding of the invasion mechanisms of carnivorous gastropods from the perspective of the digestive system, since invasion ability may be closely related to ingestion and digestion.
overexploitation and threats to its breeding grounds (Yang et al., 2007; Wei et al., 1999). Since 1992, efforts have been made to establish commercial aquaculture of the species due to its economic importance (Yuan, 1992). However, large-scale aquaculture of R. venosa has been seriously restricted by difficulties in rearing larvae to the juvenile stage, due to the mass mortality of planktonic larvae, which has become a major bottleneck in R. venosa aquaculture (Yuan, 1992; Yang et al., 2007). Therefore, it is necessary to understand the larval development process and establish technical procedures for early R. venosa larval culture to avoid overexploitation. As R. venosa is a mollusc, metamorphosis is a vital developmental event in its life cycle and occurs over a short time period (generally < 48 h). Metamorphosis is associated with high mortality (Huan et al., 2015), and the early larval phase is the most vulnerable stage in the R. venosa life cycle (Harding, 2006). R. venosa population dynamics are controlled by pre-metamorphosis recruitment and post-metamorphosis survival. Competent larvae need to find a suitable attachment site for metamorphosis, and an inappropriate substrate may be fatal (Bishop et al., 2006). Extensive morphological and physiological changes, such as velum degradation and reabsorption, foot proliferation and elongation, initiation of rapid secondary shell growth, and development of the radula, occur during metamorphosis. R. venosa larvae exhibit several characteristics that differ from those of other molluscs; these include a longer metamorphosis stage (30–35 days) and a larger size (1400–1500 μm) than those of the competent larvae of most other molluscs (Wei et al., 1999; Pan et al., 2013) and transition of food habits from herbivory to carnivory. This transition does not occur in lifelong herbivorous gastropods and is crucial for the metamorphosis of R. venosa larvae. In our previous study, we found that the mass mortality of larvae and juveniles mainly resulted from the lack of or delay in appropriate feeding, which can result in cannibalism and failure of metamorphosis (Yu et al., 2018). Therefore, appropriate feeding at the appropriate time can efficiently prevent mortality caused by cannibalism and metamorphosis failure. Thus, to establish technical procedures for early R. venosa larval culture, research on the development of the digestive system is required. Successful digestive system development is significant for the survival and growth of larvae because an efficient digestive system enables the larvae to capture, ingest, digest and absorb food (Kjorsvik et al., 2004). Thus, morphological and functional changes in the digestive system play vital roles in the metamorphic transition of R. venosa from a diet of microalgae to one of bivalve molluscs (Pan et al., 2013). Previous studies have found that many genes and proteins associated with food intake and digestion, including some digestive enzymes, are differentially expressed between the larval and postlarval stages in R. venosa (Song et al., 2016 a), and researchers have shown that phytophagous digestive enzymes (such as cellulase) are downregulated after metamorphosis, whereas carnivorous digestive enzymes (such as trypsin) are upregulated. Ontogenetic changes in the types and concentrations of digestive enzymes, which are indicative of shifts in the ability to digest dietary components, have been used to identify dietary shifts in a range of invertebrates (Hammer and Watts, 2000; Johnston, 2003). For example, changes in digestive enzyme activity and hepatopancreatic development in Macrobrachium rosenbergii occur when individuals shift from carnivorous to omnivorous habits during the larval period (Kamarudin et al., 1994). Similarly, changes in protease and αamylase activities occur in the black tiger prawn Penaeus monodon during the transitions from herbivory in the nauplius stage to carnivory in the mysis stage and then to omnivory in juveniles and adults, reflecting changes in feeding preference. Although changes in feeding preference are common in invertebrates, few studies have been conducted on the ontogenetic changes in digestive enzymes that occur in R. venosa; such studies may assist the identification of critical ages and sizes related to dietary changes in this species (Song et al., 2016 b). Most molluscs need to undergo a short or lifelong herbivorous stage, during which they feed mainly on microalgae or other plants. In this
2. Materials and methods 2.1. Larval rearing and sample preparation Larvae of R. venosa were obtained from Blue Ocean Co., Limited (Laizhou, Shandong, China). The parent culture, spawn, intramembrane larvae and planktonic larvae were cultured at a density of 0.1 ind/ml in 3 × 5 × 1.5 m cement pools at 24.6–25.6 °C according to the methods of Yang et al. (2007). Isochrysis galbana, Chlorella vulgaris and Platymonas subcordiformis were mixed and served as the diet for the planktonic larvae. The average shell height (or mean size of the intramembrane larva) and the daily increase in shell height were measured in 30 randomly selected larvae every day following the methods of Pan et al. (2013). Intramembrane larval samples were collected from 1 to 13 days after spawning (das) at seven major developmental stages: the cleavage stage (c), the blastula stage (b), the gastrula stage (g), the trochophore stage (t), the early intramembrane veliger stage (ev), the middle intramembrane veliger stage (mv), and the later intramembrane veliger stage (lv). Planktonic larval and postlarval samples were collected from 14 to 60 das at five major developmental stages: the one-spiral whorl (V–I) stage, the two-spiral whorl (VII) stage, the three-spiral whorl (V-III) stage, the four-spiral whorl (competent larva, V-IV) stage, and the juvenile (postlarva, J) stage. The samples were monitored under a microscope to ensure developmental synchrony. Whole larval samples at each development stage were taken in triplicate and octuplicate for molecular and biochemical analyses, respectively, since the individual larvae were too small and difficult to 2
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from the GenBank database, and DNAMAN software was then used to perform multiple-sequence alignments. Phylogenetic trees were constructed by MEGA7.0 using the neighbor-joining (N-J) method.
dissect, and each sample was then washed with PBS, snap-frozen in liquid nitrogen, and stored at −80 °C until use. 2.2. Extraction of total RNA
2.5. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from all the larval samples using a TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan) to clone fulllength cDNA and analyze the mRNA expression of the cellulase and trypsin genes following the manufacturer's protocol. The purity and concentration of the RNAs were measured by a Nanodrop spectrophotometer (Thermo Scientific, USA) and gel electrophoresis.
Primers for qRT-PCR were designed based on the full-length cDNA sequences of the cellulase and trypsin genes; RT-Cel-F and RT-Cel-R were used as forward and reverse primers, respectively, for the cellulase gene, and RT-Try-F and RT-Try-R were used as forward and reverse primers, respectively, for the trypsin gene. The 60S ribosomal protein L28 (RL28-F, RL28-R) was used as a housekeeping gene for signal normalization within the experiment (Song et al., 2017). First-strand cDNAs for qRT-PCR were synthesized using reverse transcriptase (TaKaRa). A SYBR Green-based real-time PCR assay (SYBR PrimeScript™ RT-PCR Kit II, TaKaRa) was used with an Eppendorf Mastercycler® ep Realplex (Eppendorf, Hamburg, Germany) for qRT-PCR analysis. Reactions containing 10 μl of Maxima™ SYBR Green qPCR master mix (TaKaRa), 10 pmol of forward or reverse primer in a volume of 0.5 μl each, 1 μl (100 ng) of cDNA and 8 μl of sterile deionized water were prepared in a total volume of 20 μl. Amplifications were performed according to the manufacturer's instructions. Standard curves were made with cDNA template diluted 10, 102, 103, 104, and 105 fold. qRTPCR was performed using the following thermocycler program: 95 °C for 5 s and 40 cycles of 95 °C for 15 s and 60 °C (cellulase) or 58 °C (trypsin) for 30 s. Relative gene expression was calculated using the 2−ΔΔCt method.
2.3. Cloning of the full-length cDNAs for the cellulase and trypsin genes 2.3.1. Cloning of the middle fragments of the full-length cDNAs Primer pairs for the middle fragments of the cellulase and trypsin genes based on expressed sequence tags in the transcriptome library of R. venosa (Song et al., 2016a,b) were designed using Primer Premier 5.0 software and synthesized by Tsingke (China). All of the primers used in this study are listed in Supplementary Table 1. First-strand cDNA was synthesized using a Prime Script™ RT Reagent Kit with gDNA Eraser (TaKaRa, Japana), and the RNA used for first-strand cDNA synthesis was a mixture of RNA samples from R. venosa specimens at all developmental stages. The middle fragments of the two genes were amplified by polymerase chain reaction (PCR) using Ex Taq DNA polymerase (TaKaRa). The PCR program implemented in a thermal cycler (TaKaRa) was as follows: initial denaturation at 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 55 °C (cellulase) or 57 °C (trypsin) for 30 s and 72 °C for 40 s and a final extension at 72 °C for 10 min. PCR products were purified using a 1% agarose gel with a 2,000 bp ladder used as a marker. Bands of the expected sizes were excised from the gel and subsequently cloned into the pMD19-T vector (TaKaRa) and sequenced by Tsingke (China), and their sequences were used to query GenBank.
2.6. Digestive enzyme activity assays Whole larval samples were collected and homogenized for biochemical analysis of the enzymes. After homogenization, each sample was centrifuged at 4 °C and 3000×g for 10 min, and the supernatant was collected and stored at −20 °C. The soluble protein content of all the samples was determined by the method using bovine serum albumin as a standard. The cellulase, trypsin, pepsin, chymotrypsin, αamylase and lipase activities were evaluated using corresponding assay kits (Nanjing Jiancheng, Bioengineering Institute, China), and the results of the enzyme assays were expressed as specific activity in units per mg protein (U/mg protein). Cellulase activity was measured using a cellulase assay kit (A138) with the 3,5-dinitrosalicylic acid (DNS) method. The chromogenic reaction was incubated in boiling water for 15 min, and the absorbance of the samples at a wavelength of 550 nm was measured. One unit of cellulase was defined as 1 mg of enzyme, which hydrolyzed the substrate and generated 1.0 μg of glucose in 1 min. Trypsin activity was detected by a trypsin assay kit (A080-2) based on ultraviolet colorimetry. Arginine ethyl was used as the substrate, and the reaction was performed at 37 °C. The absorbance of the samples at a wavelength of 253 nm was read at 30 s and 20 min 30 s to analyze trypsin activity. One unit of trypsin was defined as 1 mg of enzyme, which generated a change in optical density (OD) of 0.003 in 1 min at 37 °C. A chymotrypsin assay kit (A080-3) was used to analyze chymotrypsin activity using casein as the substrate by reading the absorbance of the samples at a wavelength of 660 nm, and 1 U of chymotrypsin was defined as 1 mg enzyme, which produced 1 μg amino acid in 1 min at 37 °C, pH 8. An α-amylase assay kit (A016-1) was used to analyze α-amylase activity based on starch-iodine colorimetry by reading the absorbance of the samples at a wavelength of 660 nm. One unit of α-amylase was defined as 1 mg of enzyme, which hydrolyzed 10.0 mg of starch in 30 min. Lipase activity was determined using a lipase assay kit (A054-2) with methyl-halogen as the substrate by continuously monitoring the absorbance of the samples at a wavelength of 570 nm for 3 min at 37 °C following the manufacturer's instructions. The rates of change in the absorbance were recorded to analyze lipase activity. Pepsin activity was determined using a pepsin assay kit (A0801) with casein as a substrate following the manufacturer's instructions.
2.3.2. Cloning of the 5′ and 3’ fragments of the full-length cDNAs First-strand cDNA (5′- and 3′-rapid amplification of cDNA ends (RACE)-ready cDNAs) synthesis and RACE were performed using a SMARTer™ RACE cDNA Amplification Kit (Clontech, USA). The primers used for RACE were designed based on the middle fragment sequences determined above using Primer Premier 5 software. The 5′ ends of the cellulase and trypsin genes were amplified with the primers try-5′GSPn and cel-5′- GSPn, respectively, and a universal primer mix (UPM), and the 5′-RACE-ready cDNAs were used as the templates. The 3′ ends of the cellulase and trypsin genes were amplified using the primers try3′- GSPn and cel-3′- GSPn, respectively, and a UPM, and the 3′RACEready cDNAs were used as the templates. PCR amplification to confirm the full-length cDNAs was conducted as follows: 94 °C for 2 min; 35 cycles of 94 °C for 30 s, 50–60 °C for 30 s, and 72 °C for 1 min; and 72 °C for 10 min (Eppendorf). PCR products were inserted into the pMD19-T vector (TaKaRa) and used to transform JM109 competent cells (TaKaRa) according to the manufacturer's instructions, and the nucleotide sequences of 10 positive clones were confirmed by Sangon Biotech (China). The sequences were analyzed and assembled by DNAStar software to obtain the full-length cDNA sequences. 2.4. Sequence and phylogenetic analyses The full-length cDNAs and protein sequences of the cellulase and trypsin genes were analyzed using the corresponding National Center for Biotechnology Information BLAST programs (http://www.ncbi.nlm. nih.gov/BLAST/). Functional sites or domains in the protein sequences were predicted using the SMART program (http://smart.emblheidelberg.de/). The molecular masses and theoretical isoelectric points of the putative proteins were predicted using ExPasy Compute pI/Mw software (http://www.expasy.org/tools/pi_tool.html). The cDNA sequences of the two genes from different species were obtained 3
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Fig. 1. Larval growth in terms of shell height (a) and daily increase in shell height (b) during R. venosa larval development for up to 60 das. Data are expressed as the mean ± SE.
The chromogenic reactions were performed at 37 °C for 20 min, and the absorbance of the samples at a wavelength of 660 nm was read to analyze pepsin activity. One unit of pepsin was defined as 1 mg of enzyme, which produced 1 μg of amino acid in 1 min at 37 °C and pH 3.
intramembrane larvae to planktonic larvae was significantly lower than the growth rate before this transition. In contrast, the growth rate was very high in planktonic larvae, especially in those in the V-II and V-IV stages.
2.7. Data analysis
3.2. Identification and analysis of the cellulase and trypsin genes
The larval growth data, qRT-PCR expression results and the biochemical enzyme activities of cellulase, trypsin, chymotrypsin, α-amylase, lipase and pepsin were analyzed by one-way ANOVA with significance indicated by P < 0.05. Tukey's test was performed to determine normality, and equality of variances was assessed by Levene's test before ANOVA was performed. All statistical analyses were preformed using SPSS 19.0 software (SPSS Inc., USA).
The full-length cDNA of the cellulase gene is 2,086 bp in length, comprising a 122-bp 5′ UTR, a 245-bp 3′ UTR, and a 1,719-bp ORF encoding 572 amino acids (GenBank accession no. MK850389) (Fig. 2 a). The calculated molecular mass and predicted isoelectric point of the cellulase protein are 63.76 kDa and 6.62, respectively. The predicted protein sequence of cellulase, a typical modularized enzyme, contains an N-terminal CBM4_9_ carbohydrate-binding domain and a C-terminal glycoside hydrolase GHF10 catalytic domain (GHF10). The full-length cDNA of the trypsin gene is 1,587 bp in length, comprising a 93-bp 5′ UTR, a 639-bp 3’ UTR, and an 855-bp ORF encoding 284 amino acids (GenBank accession no. MK850390) (Fig. 3 a). The calculated molecular mass and predicted isoelectric point of the trypsin protein are 30.58 kDa and 8.78, respectively. Domain architecture analysis indicated that the trypsin gene contains a signal peptide (amino acids 1–46) and the amino acid sequence of the trypsin-like serine protease (Tryp_SPc) family domain (amino acids 47–279).
3. Results 3.1. Larval growth The growth of R. venosa larvae was measured at 0–60 das by determining the shell height (Fig. 1 a) and calculating the daily increase in shell height (Fig. 1 b). The daily growth rate of the intramembrane larvae was low, and the growth rate during the transition from 4
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Fig. 2. Characteristics of the amino acid sequence deduced from the cellulase gene. (a) The signal peptide sequence (1–20) and the amino acid sequences of the CBM4_9_ (21–150) and GHF10 (258–507) domains are denoted by red, blue and green boxes, respectively, and the active site amino acids are denoted by green triangles. (b) Multiple alignment of amino acid sequences deduced by cellulase genes. Pomacea canaliculate [PVD26373.1], Branchiostoma belcheri [XP_019626719.1], Paenibacillus ihumii [WP_055105560.1], Triticum urartu [EMS67873.1], Brachypodium distachyon [XP_003560192.1], Lingula anatina [XP_013410933.2]. (c) Phylogenetic tree based on the amino acid sequences of cellulase proteins from R. venosa and other species. The tree was constructed based on multiple sequences generated by Clustal X and aligned using the N-J method in MEGA 7.0. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3. Homology and phylogenetic analyses of the cellulase and trypsin genes
used for homology and phylogenetic analyses. A BLAST search showed that the predicted amino acid sequence of cellulase from R. venosa shared the highest sequence identity with cellulase from Pomacea
Cellulase and trypsin protein sequences from different species were 5
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Fig. 2. (continued)
(WP_055105560.1). Compared with the sequence of cellulase, the sequence of trypsin from R. venosa shared low similarity with the trypsin sequences of other species. The sequence identity of trypsin from R. venosa with trypsin sequences from P. canaliculata (PVD20786.1),
canaliculata (XP_025105693.1, 59.64% identity), followed by cellulases from Branchiostoma belcheri (XP_019626719.1, 41.56% identity), Lingula anatine (XP_013410933.2, 35.85% identity), Brachypodium distachyon (XP_019627090.1, 33.58) and Paenibacillus ihumii 6
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Fig. 3. Characteristics of the amino acid sequence deduced from the trypsin gene. (a) The signal peptide (amino acids 1–46) and the amino acid sequence of the Tryp_SPc domain (amino acids 46–283) are denoted by red and blue boxes, respectively, and the signal peptide breaking site, substrate binding site and active site are denoted by green, blue and red triangles, respectively. (b) Multiple alignment of amino acid sequences deduced from trypsin genes. Pomacea_canaliculata [PVD36883.1], Biomphalaria_glabrata [XP_013088769.1], Drosophila_bipectinata [XP_017105525.1], Pelodiscus_sinensis [XP_006120382.1], Zonotrichia_albicollis [XP_026654917.1], Bos_taurus [DAA30409.1], Mus_ musculus [NP_001019869.1]. (c) Phylogenetic tree based on trypsin amino acid sequences from R. venosa and other species. The tree was constructed based on multiple sequences generated by Clustal X and aligned using the N-J method in MEGA 7.0. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
cellulase and trypsin genes in R. venosa at different developmental stages. The cellulase gene showed very low expression during the early developmental stages, and a significant increase in cellulase gene expression occurred from the early larval stage to the planktonic larval stage (P < 0.05); a high level of cellulase gene expression was maintained throughout the latter stage (Fig. 4 a). However, a significant decrease in cellulase gene expression occurred at the postlarval stage. Trypsin mRNA levels were detectable during the early stages of development; trypsin expression was high in the c stage and then gradually decreased with larval growth. Trypsin mRNA was expressed at low levels in the whole planktonic larval stage, whereas significantly (P < 0.05) high trypsin mRNA expression levels, almost identical to those at the c stage, were observed in the postlarval stage (Fig. 4 b).
Pocillopora damicornis (XP_027047043.1), Parasteatoda tepidariorum (XP_021001494.1), Xiphophorus maculatus (XP_023205443.1), Penaeus vannamei (ROT79329.1) and Trichuris suis (KHJ42080.1) was 41.15%, 39.84%, 39.18%, 37.55%, 35.25% and 34.71%, respectively. The deduced cellulase and trypsin protein sequences were used for multiplesequence alignments using the ClustalW sequence alignment program (Figs. 2 b and Fig. 3 b). To further clarify the evolutionary relationships between the R. venosa cellulase and trypsin genes and those of other species, we selected > 20 species for phylogenetic analyses and constructed phylogenetic trees by the N-J method. In the phylogenetic tree for the cellulase genes, R. venosa cellulase clusters with the cellulase genes from P. canaliculata and Ampullaria crossean (Fig. 2 c). Trypsin from R. venosa clusters with the trypsin genes from P. canaliculata and Lottia gigantea, showing low similarity with trypsin genes from other species (Fig. 3 c). The cellulase and trypsin genes from R. venosa both belong to the Mollusca cluster and are closely related to those of other gastropods.
3.5. Digestive enzyme activities In the present study, enzyme activities were detectable at as early as the hatching stage. Low levels of cellulase activity were observed during the intramembrane larval stages, and the levels increased from the V–I stage, with a high level of cellulase activity maintained throughout the planktonic larval stage. Cellulase activity peaked at the
3.4. Digestive enzyme gene expression determined by qRT-PCR qRT-PCR was performed to detect the mRNA expression levels of the 7
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Fig. 3. (continued)
the V–I larval stage, with the level subsequently maintained throughout the planktonic larval stage (P < 0.05). A large increase in trypsin activity occurred in the postlarval stage, and trypsin activity showed peak levels throughout the larval stage (P < 0.05) (Fig. 5 b).
V-III stage and then decreased, with a significant decrease in cellulase activity observed in the postlarval stage (P < 0.05); cellulase activity was lowest in the whole larval stage (Fig. 5 a). Low trypsin activity was observed at early developmental stages and significantly increased in 8
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Fig. 4. qRT-PCR analysis of cellulase and trypsin mRNA expression during R. venosa larval development (mean ± SE, n = 3). Different superscripted letters indicate significant differences (P < 0.05).
(Icely and Nott, 1992; Ceccaldi, 1997; Jones et al., 1997a; 1997b). For instance, high protease activity indicates protein catabolism, and cellulase activity suggests carbohydrate (i.e., starch and glycogen) catabolism (Kamarudin et al., 1994; Rodriguez et al., 1994; Johnston, 2003). The timing and quantity of digestive enzyme secretion reflect the development of the digestive system. Additionally, the larval feeding pattern is directly related to the ontogeny of digestive enzymes. Digestive enzymes in aquatic animals, especially fish and crustaceans, have been widely researched, whereas relatively little research has been conducted on digestive enzymes in molluscs (Yang et al., 1998), and few studies have focused on changes in digestive enzymes during individual development. Previous studies have indicated that significant changes in digestive enzymes occur during development in abalone and B. areolata (Bansemer et al., 2016; Jiang et al., 2012; Wei et al., 2007). Therefore, digestive enzymes might be useful as indicators of the early
In addition to the cellulase and trypsin activities, the activities of chymotrypsin, α-amylase, lipase and pepsin were detected. The chymotrypsin and pepsin activities ranged from 2 to 10 U mg−1 protein, whereas the activity of α-amylase ranged from 0.2 to 1 U mg−1 protein and that of lipase ranged from 0.04 to 0.08 U mg−1 protein. Although the activity of α-amylase increased significantly before the planktonic larval stage and peaked at the lv stage, all four digestive enzymes were active at low levels throughout the early development stage.
4. Discussion Changes in digestive enzymes during the early development stage can provide useful information about individual development because digestive enzymes are responsible for the breakdown of nutrient reserves to provide energy for maintenance, development and growth 9
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Fig. 5. Specific (a) cellulase, (b) trypsin, (c) chymotrypsin, (d) α-amylase, (e) lipase and (f) pepsin activities during the development of larval R. venosa. (means ± SEs, n = 8). Different superscripted letters indicate significant differences (P < 0.05).
canaliculata (Imjongjirak et al., 2008). These differences in the GHF subfamily suggest that the cellulase proteins in gastropods might have separated from those in other molluscs during evolution. In contrast, the full-length cDNA sequence of trypsin from R. venosa showed low degrees of sequence homology with those from other animals (< 50%) and showed the highest degree of sequence similarity with a hypothetical protein from P. canaliculata (41.15%). The Tryp_SPc family is ubiquitous in animals and plays diverse roles, especially in the digestive system (Wu et al., 2009). However, there has been little research on trypsin in molluscs. The trypsin cloned from R. venosa may be a new type of trypsin-like serine protease in molluscs. As in most gastropods (Liu and Sun, 2008; Zhou et al., 1996; Zhou et al., 2000), in R. venosa, early development comprises three stages: an intramembrane larval stage, a planktonic larval stage and a postlarval stage. Digestive enzymes differ significantly depending on the developmental stage, especially at the critical points of different developmental stages. Throughout the intramembrane larval stage, R. venosa
development and metamorphosis of gastropods that undergo food habit transition. To further understand the development of the digestive system in the early development and metamorphosis of R. venosa, we investigated changes in cellulase and trypsin mRNA expression and bioactivity during R. venosa growth. In the present study, the full-length cDNA sequences of the cellulase and trypsin genes were cloned from R. venosa, and cDNA libraries were generated from samples containing R. venosa larvae at different developmental stages. Cellulase from R. venosa showed high degrees of sequence homology with cellulases from other molluscs, particularly P. canaliculata. Cellulase from R. venosa contains a catalytic domain that is a conserved 250-amino acid region that belongs to the GHF10 subfamily, whereas most reported cellulases from animals contain a catalytic domain belonging to the GHF9, GHF5 or GHF45 subfamily (Byrne et al., 1999; Yan et al., 1998; Xu et al., 2001). GHF10, a multifunctional cellulase generally involved in the microbial degradation of cellulose and xylans, was first isolated from the animals A. crossean and P. 10
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important interim period during which the expression of different kinds of digestive enzymes is regulated (Song et al., 2016 b), of which cellulase and trypsin can serve as major indicators. During food habit transition, larvae spend their energy on not only the further development of digestive organs but also velum degradation and reabsorption, foot proliferation and elongation, initiation of rapid secondary shell growth, development of the radula, and the search for suitable attachment sites. Thus, the larval growth rate decreases significantly during this transition. A previous study indicated that large-molecule protein feeding is required for the development of pancreatic digestive functions in rats (Kinouchi et al., 2012). Therefore, it may be very important to provide suitable and adequate animal bait as early as possible in the V-IV stage (competent larva) for the larvae to undergo changes in their digestive systems, metamorphosis, and further growth and development.
larval growth increased steadily and slowly, beginning at 1 das, which is consistent with the growth patterns of other gastropod larvae (Oyarzun and Strathmann, 2011). R. venosa larval growth was extremely slow during the transition from the intramembrane larval stage to the planktonic larval stage, and we speculate that the larvae need large amounts of energy to complete the hatching process. Correspondingly, both cellulase and trypsin activities were low during this stage because the larvae do not ingest any food and are wholly reliant on endogenous reserves, which is also the case for other gastropods (Zhao et al., 2012; Oyarzun and Strathmann, 2011). However, in the present study, the mRNA expression of cellulase was greatly increased beginning at the ev stage and peaked before hatching (P < 0.05), so we speculate that the digestive organs may begin to appear at this stage. Larval growth is dependent upon the successful development of the digestive system during the most critical first feeding phase (Mir et al., 2018), so development of the digestive system during the intramembrane larval stage is significant for growth during the early development stage. Molluscs are biphasic, and most of them need go through a planktonic larval period after hatching (Huan et al., 2015). During this period, the larvae mainly feed on microalgae, such as the diatoms I. galbana, C. vulgaris and P. subcordiformis, which contain plentiful cellulose. Cellulose is mainly degraded by cellulase endogenous to the mollusc digestive system. Therefore, the increase in cellulase mRNA expression before hatching may suggest that the initial development of the digestive system is complete and that the larvae are capable of digesting microalgae for planktonic life. Relative to the change in cellulase mRNA expression, the change in cellulase activity was delayed until the V–I stage. We speculate that this asynchrony may have been due to the stimulation of digestive enzyme activity by food. (Ou and Liu, 2007; Genodepa et al., 2018). Therefore, adequate and appropriate microalgal feed should be maintained in the hatching tanks of larvae to ensure abundant food upon hatching. In addition to being necessary for larval growth and development, abundant food can promote further development of the larval digestive system. Throughout the planktonic larval stage, the larval growth rate was significantly higher than the growth rate of the intramembrane larvae, whereas a decrease in the growth rate was observed at the postlarval J stage. Meanwhile, mRNA expression and cellulase activity were maintained at high levels, which reflected the microalgal diet. Additionally, a significant increase in trypsin activity was observed at the V–I stage because protein is necessary for animal development; microalgae contain large amounts of plant proteins in addition to carbohydrates. At the end of the gastropod planktonic larval stage, gastropod larvae need to undergo metamorphosis to enter the postlarval stage and develop into juveniles. A previous study indicated that the mRNA expression of cellulase and three other polysaccharide lyase genes in Haliotis discus hannai Ino was significantly increased from the early peristomial shell larval stage, which is consistent with the development of the digestive system and the appearance of new digestive organs during metamorphosis (He, 2011). However, a substantial decrease in cellulase expression was observed during B. areolata metamorphosis (Wei et al., 2007). The reason for this difference is that abalone is herbivorous, whereas B. areolata is carnivorous. In the present study, the mRNA expression of cellulase was slightly decreased in the V-IV stage (competent larvae), and the mRNA expression and activity of cellulase reached very low levels in the J stage (postlarva), consistent with a previous study on B areolata (Wei et al., 2007). In contrast, the mRNA expression and activity of trypsin remained low in the V-IV stage (competent larva) but greatly increased when the larvae underwent metamorphic transition. Relative to the cellulase and trypsin activities, the activities of the other four digestive enzymes in this process remained very low, with no pronounced changes in activity. This finding supports the conjecture in previous research that changes in the regulation of these digestive enzymes are responsible for the change in diet after metamorphosis and that the V-IV stage (competent larvae) is an
5. Conclusion Although the mRNA expression of cellulase increased beginning at the ev stage, a significant increase in cellulase activity was observed at the V–I stage. This significant increase suggests that the initial development of the digestive system is complete at this stage and that the larvae are capable of digesting microalgae for planktonic life. Additionally, the mRNA expression and activity of trypsin were greatly increased in the J stage (postlarva), whereas those of cellulase were decreased, which reflected the food habit transition from herbivory to carnivory. The activities of cellulase and trypsin were 10 to 104 times greater than those of the other four digestive enzymes studied. This finding suggests that cellulase and trypsin are the major digestive enzymes in R. venosa and can serve as indicators for the development of the digestive system and food habit transition during the early development of R. venosa. Based on our results, we suggest that animal bait be provided as early as possible in the V-IV stage to promote the development of R. venosa and ensure successful metamorphosis. Integrative studies on the histological organization of digestive structures and histochemical localization studies are needed to completely understand digestive development in R. venosa.
Declaration of competing interest The authors report no conflict of interest.
Acknowledgments The authors thank the Ma Shan Group Co., Ltd., for providing support with the experimental material. This research was supported by the National Natural Science Foundation of China (grant no. 31572636), the Natural Science Foundation of Shandong Province (grant no. ZR2019BD003), the China Postdoctoral Science Foundation (grant no. 2019M652498), the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-49), the Industry Leading Talents Project of Taishan Scholars (Recipient: Tao Zhang), the ‘Double Hundred’ Blue Industry Leader Team of Yantai (Recipient: Tao Zhang), and the Creative Team Project of the Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology (no. LMEES-CTSP-2018-1). The funders had no role in the study design, data collection or analysis, decision to publish or preparation of the manuscript.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734722. 11
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