Cloning and characterization of tyrosine decarboxylase (TDC) from Litopenaeus vannamei, and its roles in biogenic amines synthesis, immune regulation, and resistance to Vibrio alginolyticus by RNA interference (2023)


It is well known that the biogenic amine fulfills multifunctional roles as a neurotransmitter, neuromodulator, and neurohormone in both vertebrate and invertebrate organisms (Roeder, 1999). The synthesis of biogenic amine is divided into two pathways and tyrosine (TR) is the metabolic precursor of both. One is norepinephrine (NE) synthesis pathway (TR → l-3,4-dihydroxyphenylalanine (l-DOPA) → dopamine (DA) →NE), and the other is octopamine (OA) synthesis pathway (TR → tyramine (TA) →OA) (Chase and Koelle, 2007). The metabolic enzymes participating in the NE synthesis pathway are (1) tyrosine hydroxylase (TH) hydroxylates TR to l-DOPA; (2) DOPA decarboxylase (DDC) decarboxylates l-DOPA to DA; and (3) dopamine beta-hydroxylase (DBH) hydroxylates DA to NE (Cheng et al., 2017; Mapanao and Cheng, 2016; Lin et al., 2020). On the other hand, the metabolic enzymes participating in the OA synthesis pathway are (1) tyrosine decarboxylase (TDC) decarboxylates TR to TA; and (2) tyramine beta-hydroxylase (TBH) hydroxylates TA to OA (Alkema et al., 2005).

The effects of biogenic amines on the physiological regulatory mechanisms of molting, reproduction, osmoregulation, carbohydrate metabolism, frequency of the heartbeat, and the dilation and constriction of the stomach within a wide range of crustacean species have been evaluated through pharmacological, biochemical and molecular-genetic analysis (Mattson and Spaziani, 1985; Howard and Talbot, 1992; Yeh et al., 2006; Lüschen et al., 1993). At the biological cellular level, biogenic amines activate the specific receptor proteins, which belong to a family of G-protein coupled receptors (GPCR). Activation of these receptors leads to alterations in the level of intracellular second messengers, including cyclic adenosine monophosphate (cAMP) and calcium, which initiates the downstream cascade to regulate many complex processes (Hauser et al., 2006; Liu et al., 2019).

Most of the biogenic amine-related research has focused on physiological regulation and behaviors in crustaceans (Glanzman and Krasne, 1983). However, we have demonstrated effects of biogenic amine on immunity of Litopenaeus vannamei, Penaeus monodon, and Macrobrachium rosenbergii. Interestingly, the biogenic amines in the NE synthesis pathway downregulate the immune response of shrimp (Cheng et al., 2005; Mapanao et al., 2018a, Mapanao et al., 2018b; Yeh et al., 2006), and in the OA synthesis pathway, the immune response is upregulated (Kuo et al., 2019a, 2019b). We also cloned and characterized the metabolic enzymes in the NE synthesis pathway of L. vannamei. Through the gene silencing or enzyme inhibitor of TH, DDC, and DBH, it confirmed the function of these enzymes in biogenic amine synthesis and immune regulation of L. vannamei (Lin et al., 2020; Cheng et al., 2017). However, the enzymes in the OA synthesis pathway, TDC and TBH, had not been tested in L. vannamei.

Owing to lack of adaptive immunity, shrimp rely on innate immunity to defend the foreign particle or microbial organism. Shrimp haemocytes, which can be divided into three types including the granulocyte, the semi-granulocyte, and the hyaline cell are considered to have critical roles in shrimp immunity. Haemocytes originated from hematopoietic tissues and are regulated during molting, seasonal variation, infection or other stress (Söderhäll et al., 2016). The granulocyte and semi-granulocyte store the components of the prophenoloxidase-activating (proPO) system which is an important immune reaction, initiating the melanization cascade by degranulation (Cerenius et al., 2010). Haemocytes also perform cellular reactions such as phagocytosis, encapsulation, and nodule formation. Respiratory burst (RB) is a rapid procedure in reactive oxygen species production during phagocytosis, which is essential in eliminating microbes (Wang and Chen, 2005). Previous studies have demonstrated that the immune responses, including hematopoiesis, the proPO system, RB, and phagocytotic activity, were regulated by biogenic amines (Noonin, 2018; Liu et al., 2019).

L. vannamei is one of the most important aquacultural species and it is a successful species in the blue revolution (Asche et al., 2020), but the shrimp industry encountered high economic losses due to massive pathogen infection. Although TA- and OA-mediated immunological and physiological responses, and susceptibility to pathogens have been evaluated, unfortunately, the key enzyme, TDC, in the first metabolic step of the OA synthesis pathway had not been identified in L. vannamei. Therefore, the purposes of the present study were (1) to clone and characterize the full-length gene sequence and structure modeling of TDC from L. vannamei (LvTDC), (2) to detect the tissue distribution pattern of LvTDC gene expression, (3) to assess the effect of LvTDC on the susceptibility of L. vannamei to Vibrio alginolyticus through gene silencing, and (4) to determine immune and physiological responses in LvTDC-dsRNA shrimp.

Cited by (5)

  • Transcriptional and metabolomic investigation of the stress response in snow crab during simulated transport condition (Chionoecetes opilio)

    2023, Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics

    The molecular mechanisms underlying the stress response are poorly described in crustaceans. This includes the snow crab (Chionoecetes opilio), a commercially important stenotherm species distributed throughout the northern hemisphere. A better understanding of the stress response in C. opilio is desperately needed for commercial and conservation purposes. The purpose of this study was to investigate the transcriptional and metabolomic response of C. opilio exposed to stressors. Crabs were randomly assigned to 24 or 72h treatment groups where they were exposed to conditions simulating live transport (handling and air exposure). A control group was kept in cold (2°C) and well‑oxygenated saltwater. The hepatopancreas of the crabs was sampled to perform RNA-sequencing and high-performance chemical isotope labeling metabolomics. Differential gene expression analyses showed that classic crustaceans' stress markers, such as crustacean hyperglycemic hormones and heat shock proteins, were overexpressed in response to stressors. Tyrosine decarboxylase was also up-regulated in stressed crabs, suggesting an implication of the catecholamines tyramine and octopamine in the stress response. Deregulated metabolites revealed that low oxygen was an important trigger in the stress response as intermediate metabolites of the tricarboxylic acid cycle (TCA) accumulated. Lactate, which accumulated unevenly between crabs could potentially be used to predict mortality. This study provides new information on how stressors affect crustaceans and provides a basis for the development of stress markers in C. opilio.

  • Intestinal morphology and microflora to Vibrio alginolyticus in pacific white shrimp (Litopenaeus vannamei)

    2022, Fish and Shellfish Immunology

    Citation Excerpt :

    A study has proved that pvsA and pvsD genes were involved in the biosynthesis of siderophores of V. alginolyticus [18]. In recent years, researchers have focused on the cloning and identification of effective enzymes to explore the mechanism of L. vannamei infected with V. alginolyticus [19–21]. However, the intestinal response to L. vannamei infected with V. alginolyticus was not clear and needed to be further explored.

    In recent years, the shrimp farming industry encountered significant economic losses induced by Vibrio alginolyticus. In this study, the influence of Vibrio alginolyticus on intestinal histomorphology and microbiome composition in Litopenaeus vannamei were studied. The results showed that the intestinal mucosal epithelial cells of Vibrio group (VA group) injected only with Vibrio alginolyticus showed large area exfoliation at 12h, and the tissue morphology of intestine recovered at 48h. Compared with the control group (CK group), the abundance of Proteobacteria was significantly higher (P<0.05), while the abundance of Actinobacteria was significantly lower after infection with Vibrio alginolyticus. The abundance of Shewanella in intestinal microbiome of Litopenaeus vannamei was significantly higher at 12h(P<0.05), but the abundance of Candidatus_Bacilloplasma was significantly lower at 48h after infection (P<0.05). In VA group, the diversity of intestinal microbiome was significantly lower at 12h, which could be caused by the proliferation of Candidatus_Bacilloplasma and Shewanella. All above findings suggested that the stability of the dynamic balance of microbiome in the intestine helped Litopenaeus vannamei to resist pathogen colonization.

  • Cloning and characterization of a key enzyme in octopaminergic pathway: Tyramine beta-hydroxylase from Litopenaeus vannamei, as expressed during Vibrio alginolytics infection and hypothermal stress

    2021, Fish and Shellfish Immunology

    Citation Excerpt :

    The release of BAs is considered to be the primary response to physiological stress, and subsequently, the physiological and immunological regulation are the secondary responses [3,4]. Several BAs including dopamine (DA), norepinephrine (NE), tyramine (TA) and octopamine (OA) have been identified and quantitatively measured in crustaceans [5,6]. l-tyrosine is a precursor for the production of BAs, which can be metabolized to norepinephrine (NE) or octopamine (OA) via different pathways (Fig. 1).

    Tyramine beta-hydroxylase (TBH) is needed for the biosynthesis of the octopamine (OA) from tyramine (TA). Both OA and TA act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. In this study, TBH was identified in white shrimp, Litopenaeus vannamei, and further investigation on its potential function was conducted after inducing hypothermal stress and Vibrio alginolyticus infection. TBH of L. vannamei (LvTBH) was comprised 2178 nucleotide residues and contained an open reading frame encoding 408 amino acids, belonging to the Copper type II, ascorbate-dependent monooxygenases, was characterized by two Cu2_monooxygen domains and five glycosylation sites. LvTBH expression was especially abundant in muscle, and mainly in brain and thoracic ganglia of nervous system, eyestalk tissues, epithelium, and stomach, as determined by quantitative real-time PCR. The effects of hypothermal stress showed significant increases in LvTBH at 15, 30 and 60min in brain and at 30min in haemocyte, accompanied by an increase in OA level in haemolymph from 15 to 60min. Significant increases in LvTBH occurred at 15, 30 and 60min in haemocyte and at 60min in brain tissue, and was proportional to the OA level of haemolymph under Vibrio alginolyticus infection from 30 to 60min. Here, we demonstrated that LvTBH is functionally responsible for biogenic amine synthesis, suggesting that the increased release of OA in haemolymph for potential modulation of physiological and immunological responses is the consequence of the upregulated LvTBH gene expression in L. vannamei exposed to hypothermal stress and Vibrio alginolyticus infection.

  • Synbiotic combination of prebiotic, cacao pod husk pectin and probiotic, Lactobacillus plantarum, improve the immunocompetence and growth of Litopenaeus vannamei

    2021, Fish and Shellfish Immunology

    Citation Excerpt :

    Further studies on the potential bioactive compounds from synbiotics and the ways in which these compounds operate should be conducted to clarify and better understand the processes leading to the enhancement of growth performance and immunocompetence. Catecholamines, produced from the biosynthesis of octopamine [34,50,51] and NE [35,36,52], using tyrosine as a precursor has been studied in L. vannamei. In octopamine biosynthesis, tyrosine was converted to tyramine by tyrosine decarboxylase, then tyramine was converted to octopamine by tyramine β-hydroxylase, and in norepinephrine biosynthesis, l-DOPA, DA and NE were serially produced from tyrosine following the activation of tyrosine hydroxylase, DOPA decarboxylase, and dopamine beta-hydroxylase, respectively [53].

    To reach the sustainable development goals on waste recycling, cacao pod husk (CPH), produced as an agricultural waste byproduct during the cacao bean processing was applied to manufacture CPH pectin for developing the potential for diverse application in aquaculture, minimizing CPH impact to the environment and bringing benefits to the agriculture and aquaculture industries. In this study, CPH pectin (5g/kg diet) and Lactobacillus plantarum (LP; 1010cfu/kg diet) were separately introduced to the diets of Litopenaeus vannamei for a 56-day feeding trial, and two synbiotic combinations of CPH pectin and LP (CPH pectin at 5g/kg diet+LP at 107cfu/kg diet or at 1010cfu/kg diet) were also conducted. After the 56-day feeding trial, significantly elevated percent weight gain, percent length gains and feeding efficiency in L. vannamei were only observed in synbiotic combination of CPH pectin at 5g/kg diet and LP at 107cfu/kg diet treatment, and the remainder of the treatments remained consistently similar to the control. Significantly increases in total haemocyte count, granular cells, phenoloxidase activity, and respiratory bursts were observed in L. vannamei fed with synbiotics at 7–28 days of feeding, accompanied by significant promotion of phagocytic activity and clearance efficiency in response to V. alginolyticus challenge during 56 days of feeding trial. Furthermore, at the end of the 56 days of feeding trial, shrimp receiving CPH pectin and/or LP treatments showed a significantly higher survival ratio against V. alginolyticus infection and hypothermal stress. It was therefore concluded that CPH pectin or LP was confirmed as an immunostimulant for L. vannamei to trigger immunocompetence through oral administration without negative effects within 56 days of feeding trial, and the synbiotic combination of CPH pectin and LP exhibited complementary and synergistic effects on growth performance and immunocompetence in L. vannamei.

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    Shrimp lack adaptive immune systems and mainly rely on the cellular and humoral defences, involving the haemocytes (functionally analogous to vertebrate leukocytes) in non-self matter recognition, elimination, and in downstream coagulation. Furthermore, the linkage between stress-induced catecholamine (CA), a class of biogenic amines (BAs), releasing and immunological responses has been detected in shrimp. Varied isotypes of protein kinase C (PKC) regulate multiple cellular processes following their specific location and distribution within the cells, and a novel PKC identified in Litopenaeus vannamei (termed as LvnPKC) is proposed to mediate signaling transduction of immunocompetence and BA biosynthesis. In the present study, we analyzed the effects of the LvnPKC-silenced haemocytes by co-incubating with its dsRNA on the immune responses specific to prophenoloxidase (proPO) and antioxidant systems as well as phagocytic activity. In addition, the capability of haemocytes to produce BAs was assessed. The results revealed that LvnPKC-silenced haemocytes can induce interference in phenoloxidase and superoxide dismutase activities, respiratory bursts, and phagocytic activity; meanwhile, the disturbed gene expressions of proPO activating enzyme, proPOII, lipopolysaccharide- and β-1,3-glucan-binding protein, and cytosolic manganese superoxide dismutase were detected. The same deviated pattern was observed in tyrosine, dopamine, and norepinephrine levels, and in dopamine β-hydroxylase (DBH) activity and gene expressions of tyrosine hydroxylase, DOPA decarboxylase, and DBH involving in BA biosynthesis. Taken together, these results suggest that the immunocompetence and BA biosynthesis of haemocytes can be mediated via LvPKC signaling transduction, which proved the presence of a neuroendocrine-immune regulatory network in haemocytes.

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    Roles of dopamine receptors in mediating acute modulation of immunological responses in Macrobrachium rosenbergii

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    Dopamine (DA) was found to influence the immunological responses and resistance to pathogen infection in invertebrates. To clarify the possible modulation of DA through dopamine receptors (DAR) against acute environmental stress, the levels of DA, glucose and lactate in the haemolymph of Macrobrachium rosenbergii under hypo- and hyperthermal stresses were measured. The changes in immune parameters such as total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and phagocytic activity (PA) were evaluated in prawns which received DAR antagonists (SCH23390, SCH, D1 antagonist; domperidone, DOM, D2 antagonist; chlorpromazine, CH, D1+2 antagonist) followed by hypo- (15°C) and hyperthermal (34°C) stresses. In addition, pharmacological analysis of the effect DA modulation was studied in haemocytes incubated with DA and DAR antagonists. The results revealed a significant increase in haemolymph DA accompanied with upregulated levels of glucose and lactate in prawns exposed to both hypo- and hyperthermal stresses in 2h. In addition, a significant decrease in RBs per haemocyte was noted in prawns which received DAR antagonists when they exposed to hyperthermal stress for 30min. In invitro test, antagonism on RBs, SOD and GPx activity of haemocytes were further evidenced through D1, D1, D1+D2 DARs, respectively, in the meantime, no significant difference in PO activity and PA was observed among the treatment groups. These results suggest that the upregulation of DA, glucose and lactate in haemolymph might be the response to acute thermal stress for the demand of energy, and the DAR occupied by its antagonistic action impart no effect on immunological responses except RBs invivo even though the modulation mediated through D1 DAR was further evidenced in RBs, SOD and GPx activities invitro. It is therefore concluded that thermal stress mediate stress responses not only through DAR but also via diverse pathways, and DA might modulate the levels of RBs, SOD and GPx activities mainly through D1 DAR.

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    Novel protein kinase C participates catecholamine biosynthesis and immunocompetence modulation in haemocytes of Litopenaeus vannamei

    Developmental & Comparative Immunology, Volume 103, 2020, Article 103504

    The catecholamine biosynthesis is required for physiological and immunological responses against stress, and the neuroendocrine-immune regulatory network plays a crucial role in immunocompetence of shrimp. A novel protein kinase C of Litopenaeus vannamei (LvnPKC) is involved in immune defense and signaling transduction in haemocytes, and in the present study, the gene silence technique is conducted to identify the role of LvnPKC on catecholamine biosynthesis and immunocompetence modulation in haemocytes of L. vannamei. The results show that tyrosine significantly increases in haemocytes of LvnPKC-silenced shrimp, and in the meantime, the obvious decrease of L-3, 4-dihydroxyphenylalanine and increase of dopamine as well as the consistent norepinephrine levels are detected. Tyrosine hydroxylase and dopamine β-hydroxylase activities are significantly reduced in haemocytes of LvnPKC-silenced shrimp. Total haemocyte count, hyaline cells and granulocytes insignificantly differ among treatments, and the obvious increase of phenoloxidase activity, respiratory bursts, superoxide dismutase and glutathione peroxidase activities are observed in haemocytes of LvnPKC-silenced shrimp, and furthermore, the downregulated phagocytic activity was observed. It is therefore concluded that the LvnPKC mediates catecholamine biosynthesis and immunocompetence in haemocytes, and plays a crucial role in the neuroendocrine-immune regulatory network.

© 2021 Elsevier Ltd. All rights reserved.

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