Elsevier

Fish & Shellfish Immunology

Volume 62, March 2017, Pages 31-40
Fish & Shellfish Immunology

Full length article
Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress

https://doi.org/10.1016/j.fsi.2017.01.012Get rights and content

Highlights

  • A chitinase-4 gene was cloned from P. monodon by RACE method.

  • The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp ORF.

  • qRT-PCR was performed to study the PmChi-4 expression pattern in different tissues and larvae stages.

  • PmChi-4 expression level increased after S. agalactiae and V. harveyi infection.

  • PmChi-4 expression level declined after ammonia nitrogen stress treatment.

Abstract

Chitinase is a multi-gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase-4 gene (PmChi-4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi-4 protein was 67.7 kDa and shared 61%–88% identity with the type of Chi-4s from other crustaceans. Quantitative real-time (qRT-PCR) analysis indicated that PmChi-4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi-4 was expressed throughout the whole larvae stages, and the highest level of PmChi-4 transcripts was detected at Mysis3 stage, which indicated that PmChi-4 may be involved in larval metamorphosis. In order to know whether PmChi-4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi-4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi-4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi-4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon.

Introduction

The chitinases have various functions in a wide range of organisms, which are associated with morphogenesis of arthropods, turnover of chitinous structures, nutrition digestion, and disease resistance, and the molting cycle of invertebrates [1], [2]. In crustaceans, chitin is an important component of the cuticular exoskeleton and the peritrophic matrix (PM), and function as a mechanical support and an attachment framework for the musculature [3], [4]. Due to the presence of chitin and sclerotized proteins, the cuticles of crustaceans do not possess the capacity to keep pace with body growth. Therefore, crustaceans have to degrade the old exoskeleton periodically and synthesize a new exoskeleton to match the new body size during molting (ecdysis) [5], [6], [7]. During the premolt period, the epidermis secretes chitinases to degrade the inner layers of the old exoskeleton and synthesizing a new exoskeleton at the same time [6]. Besides, the digestive tract secretes chitinases to degrade ingested chitin, modify the intestinal peritrophic membrane, and defense against viral pathogens [8], [9], [10], [11]. Due to the importance of chitinases in growth, development and immunity, more and more studies related to the chitinases were carried out. Since the first full-length cDNA of an insect chitinase gene was cloned from Manduca sexta, and the corresponding gene in the M. sexta genome was characterized [12], many cDNAs encoding chitinases and chitinase-like proteins from insect species (e.g., Drosophila melanogaster [13], Anopheles gambiae [14]) were reported in succession. According to the results of previous studies, insect chitinase genes could be classified into eight groups. Research shows that the group I and II chitinase genes are involved in insect molting and egg hatching in Tribolium castaneum, whereas the group III chitinase genes have morphogenetic roles in regulating abdominal contraction and wing expansion and the group V chitinase genes are necessary for adult eclosion [15]. However, the functions of members of the other four groups (i.e., group IV, VI, VII and VIII) are not completely clear. Although many chitinases and chitinase-like proteins have been isolated and characterized in insect species, there are few related reports in crustacean, especially in shrimps. The crustacean chitinase genes MjChi-1, MjChi-2 and MjChi-3 were first isolated and characterized from Marsupenaeus japonicus. The MjChi-1 and MjChi-3 were highly expressed in hepatopancreas compared to other tissues and the expression levels did not change significantly between the intermolt and premolt stages, while the MjChi-2 was expressed in tail fan and blade rather than hepatopancreas and the expression level of MjChi-2 was up-regulated in the tail fan and blade during the premolt stages [8], [16], [17]. Besides, six chitinase cDNAs (LvCht1 to Cht6) were identified from Litopenaeus vannamei [18] and five chitinase genes were identified from Eriocheir sinensis [19], all these results indicated that though there are many chitinase-like genes widely expressed in crustacean, the biological function or the biochemical properties of crustacean chitinases are not well established.

The chitinase-4s gene (Chi-4s) as one of the important chitinase gene have been isolated from L. vannamei, P. japonica, E. sinensis and M. nipponense, and are predominantly expressed in the hepatopancreas. Previous studies have speculated that may be involved in the turnover of the peritrophic membrane in the intestine, digestion of chitinous food and disintegrate the chitin exoskeleton during the molting cycle. But, the function of Chi-4s have not been demonstrated. In addition, high expression level of Chi-4s were detected in hepatopancreas, which are the main tissue involved in the immune response, and the major site for the synthesis of immune defense molecules involved in eliminating pathogens or other particulate matter in crustaceans [20].

Black tiger shrimp as one of three most popular cultured shrimps in the world, is also the important traditional farming shrimp in China. However, with the development of intensive culture and the ecologic environmental deterioration, various diseases caused by pathogens and environmental stresses have blossomed within booming in cultured shrimp populations, causing economic losses to commercial shrimp aquaculture [21], [22]. Previous studies have demonstrated that the suboptimal environmental conditions, such as ammonia nitrogen stress could affect the growth and molting [23].

Vibriosis is the most predominant bacterial disease, which causes mass mortalities of cultured shrimp worldwide [24]. Vibriosis in giant tiger shrimp is commonly caused by several different vibrios such as Vibrio harveyi (V. harveyi), Vibrio parahaemolyticus, and Vibrio alginolyticus. Among them, V. harveyi is the most virulent and prevalent pathogen of larval and grow-out shrimp culture [24], [25], [26], [27]. Luminous bacterial vibriosis was named after the luminous symptoms (the shrimp fluoresce) [25] and can cause severe damage to the hepatopancreas [27]. Streptococcus agalactiae is a common aquaculture pathogen, Gram-positive bacterium that is emerging globally in different fish species, such as tilapia (Oreochromis niloticus), gulf killifish (Fundulus grandis), golden pompano (Trachinotus blochii), silver pomfret (Fundulus grandis), barcoo grunter (Scortum barcoo) and giant queensland grouper (Epinephelus lanceolatus) [28], [29], [30]. Besides, the ammonia belongs to one of the major toxicant resulting from the excreta of cultured shrimp or mineralization of organic wastes like feces or excess feed, and metabolic waste products. The ammonia usually occurs in two forms: unionized (NH3) and ionized (NH4+), and the unionized form is considered more toxic to aquatic animals as it can easily diffuse across gill membranes [31]. No matter V. harveyi and S. agalactiae infection or ambient ammonia nitrogen stress can lead to high shrimp mortality and great economic loss to the aquaculture industry annually. However, up to now, no studies have studied the expression patterns of chitinases after injected with V. harveyi and S. agalactiae infection or ambient ammonia nitrogen stress in crustaceans. Therefore, in this study, we want to investigate the time-course expression profiles of PmChi-4 in response to V. harveyi and S. agalactiae infection or ambient ammonia nitrogen stress.

In this study, we described the cDNA cloning of a chitinase gene from P. monodon, which we termed PmChi-4. Phylogenetic analysis and real-time RT-PCR were conducted in different tissues at different molt stages, different development stages of larvae, and in response to V. harveyi and S. agalactiae infection or ambient ammonia nitrogen stress. All the results indicated that the PmChi-4 plays an important role in regulating shrimp molting, immunity and stress response.

Section snippets

Experimental animals

Healthy black tiger shrimps, P. monodon weighing an average of 21 ± 1 g were obtained from the experimental base of South China Sea Fisheries Research Institute in Shenzhen (Guangdong, China). The shrimps were maintained at 25 ± 1 °C in tanks containing aerated filtered seawater (31 ppt), with 50% renewed daily. After acclimatization for 7 days, the shrimp were used for experiments. The shrimps were fed with commercial diets during acclimation until 24 h before treatment.

Molt staging was

cDNA cloning and characterization of PmChi-4 gene

The full-length cDNA sequences of PmChi-4 were assembled from the three overlapping cDNA fragments (EST, 5′ and 3′ nested PCR products). The full-length cDNA of PmChi-4 was 2178 bp long, including an ORF of 1815 bp that encodes a putative protein of 604 amino acids, a 5′-UTR of 35 bp and a 3′-UTR of 328 bp with a poly A tail (Fig. 1). The predicted PmChi-4 protein is 67.7 kDa with the theoretical isoelectric point of 4.43.

Multiple sequence alignment and phylogenetic analysis

Sequence analysis with the BLASTP program revealed that the deduced amino

Discussions

Chitinase plays important physiological roles in crustaceans, including the degradation of chitinous cuticle and digestion of chitin-containing food, and defense against viral pathogens. There are only a few reports about Chi-4 in crustaceans. The information about the cloning of Chi-4 was reported only from L. vannamei [18], P. japonica [37], M. nipponense [38] and E. sinensis [19]. In this study, we had cloned and characterized a Chi-4 gene from black tiger shrimp, P. monodon. The full-length

Acknowledgements

This study was funded by China Agriculture Research System (CARS-47); Guangdong Province Project of China (2014B020202003); Guangdong Oceanic and Fisheries Project of China (A201501A06); Shenzhen Biological Industry Development Project of China (NYSW201400331010053).

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