Yongjie Liu

Identification of ecdysteroid signaling late-response genes from different tissues of the Pacific white shrimp, Litopenaeus vannamei.

Ecdysteroids initiate signaling along multiple pathways that regulate various aspects of development, maturation, and reproduction in arthropods. This study was carried out to seek the late target genes of ecdysteroid signaling from different tissues of the Pacific white shrimp, Litopenaeus vannamei. In the present study, eight isoforms of ecdysteroid receptor (EcR), two isoforms of retinoic acid X receptor (RXR), and one homolog of E75 were characterized from L. vannamei. The overall protein sequences and specific functional sites of EcR, RXR and E75 among crustacean species were found highly conserved. Tissue-specific, development stage-specific, and molt stage-specific expression patterns of LvEcR, LvRXR, and LvE75 were detected by qPCR. Double stranded RNA (dsRNA)-mediated RNA interference (RNAi) of any one of the three genes LvEcR, LvRXR and LvE75 caused specific expression changes of the other two, and resulted in corresponding expression changes of two molting related genes Cathepsin-L (LvCHSL) and Hemocyanin (LvHCyn) in the hepatopancreas, two chitin metabolism related genes chitin synthase (LvChS) and chitinase isoenzyme (LvChi2) in the epidermis, and two muscle growth related genes LvActin and myosin heavy chain (LvMHC) in the muscle. In correspondence, after in vivo injections of 20 hydroxyecdysone, specific expression changes of LvEcR, LvRXR, LvE75, LvCHSL and LvHCyn in the hepatopancreas, LvEcR, LvRXR, LvE75, LvChS and LvChi2 in the epidermis, and LvEcR, LvRXR, LvE75, LvActin and LvMHC in the muscle were also observed, respectively. Results in our study indicate multiple functions of ecdysteroids signaling in L. vannamei and the function may be time- and space-specific; ecdysteroids may act through different pathways via its functional receptor heterodimer EcR-RXR and the early responsive gene E75 to perform specific regulation roles on the target genes in different shrimp tissues; LvCHSL and LvHCyn in the hepatopancreas, LvChS and LvChi2 in the epidermis, and LvActin and LvMHC in the muscle are potential targets for ecdysteroid control. This is the first report on nuclear receptors in the economically important shrimp L. vannamei.

cDNA cloning and expression analysis of myostatin/GDF11 in shrimp, Litopenaeus vannamei

Myostatin (MSTN) and growth differentiation factor-11 (GDF11) are closely related proteins belonging to the transforming growth factor-β (TGF-β) superfamily. In vertebrates, MSTN is known to negatively regulate skeletal muscle growth, and GDF11 is found to inhibit neurogenesis. In invertebrates, only one ortholog of vertebrate MSTN and GDF11 (MSTN/GDF11) existed. Little attention has been paid on its role to date. In this study, the cDNA that encodes a 422-amino-acid MSTN/GDF11 protein (LvMSTN/GDF11) was characterized from a crustacean species, the Pacific white shrimp (Litopenaeus vannamei). Sequence analysis revealed that the overall protein sequence and specific functional sites of LvMSTN/GDF11 were highly conserved with those in other crustacean species. Expression analysis by quantitative real-time reverse transcription polymerase chain reaction technique demonstrated its tissue-specific, larval developmental stage-specific, and molt stage-specific expression pattern, respectively. After in vivo injections of 20 hydroxyecdysone (20E), LvMSTN/GDF11 transcripts were declined in the abdominal (A) and pleopod (P1) muscles, increased in the pereiopod (P2) muscle, and not affected in the thoracic (T) muscle. The observed expression profiles suggest multiple functions of LvMSTN/GDF11 in L. vannamei and its role differs during the larval development and natural molt cycle. The different responses of LvMSTN/GDF11 to acute increases of 20E in the A, P1, P2 and T muscles may indicate that LvMSTN/GDF11 is transcriptionally regulated via ecdysteroids to coincide with its specific roles in the former three muscles, while its role may be independent of 20E regulation in the T muscle.

A galectin from shrimp Litopenaeus vannamei is involved in immune recognition and bacteria phagocytosis.

Galectins are conserved family members with β-galactosides affinity that play multiple functions in embryogenesis, development and regulation of innate and adaptive immunity. However, little functional studies were reported in crustaceans. Here, a shrimp Litopenaeus vannamei galectin (LvGal) cDNA was identified with an open reading frame of 1017 bp, which encodes a putative protein of 338 amino acids. A carbohydrate recognition domain (CRD) and several amino acids residues involved in dimerization were found in LvGal. LvGal mRNA was mainly expressed in gills and hemocytes and upregulated post Vibrio anguillarum challenge. Recombinant LvGal (rLvGal) was expressed in Escherichia coli BL21 (DE3) and the purified rLvGal could strongly bind G(-) bacteria V.xa0anguillarum and G(+) bacteria Micrococcus lysodeikticus. Besides, rLvGal exhibited strong activity to agglutinate V.xa0anguillarum and weak activity to agglutinate M.xa0lysodeikticus but no obvious antibacterial activity was found with selected bacteria. In addition, inxa0vivo experiments showed rLvGal could promote phagocytosis of bacteria by hemocytes. Thus, through these collective data we predicted LvGal is involved in immune recognition and functions as a potential pattern recognition receptor.

RNAi knock-down of shrimp Litopenaeus vannamei Toll gene and immune deficiency gene reveals their difference in regulating antimicrobial peptides transcription.

NF-κB dependent antimicrobial peptides (AMPs) are of critical importance in protecting insects or mammals from microorganisms infection. However, we still do not make clear signaling pathways in regulating AMPs expression in shrimps. In this study, RNAi approach was used to study differences between Toll signaling pathway and immune deficiency signaling pathway in regulating the transcription of NF-κB dependent AMPs post bacteria challenge. Results showed that the transcription level of anti-lipopolysaccharide factor was highly suppressed in Litopenaeus vannamei immune deficiency (LvIMD) silenced shrimps by gene specific dsRNA compared to Litopenaeus vannamei Toll (LvToll) silenced shrimps with or without Vibrio anguillarum and Micrococcus lysodeikticus challenge. Conversely the transcription level of penaeidin3a was significantly suppressed in LvToll silenced shrimps compared to LvIMD silenced shrimps. However, no obvious difference was found in regulating the transcription of CrustinP. Meanwhile, we found that silencing LvToll both down regulated the transcription of Dorsal and Relish while silencing LvIMD only down regulated the transcription of Relish. At last, shrimp survival experiment showed that post V. anguillarum challenge high mortality was found both in LvToll and LvIMD silenced groups while post M. lysodeikticus challenge we saw high mortality only in LvToll silenced group. Hence, we conclude that shrimp L. vannamei Toll pathway and IMD pathway might be different in regulating the transcription of NF-κB dependent AMPs and responding to bacteria challenge but not independent of each other.

Identification and functional studies of Akirin, a potential positive nuclear factor of NF-κB signaling pathways in the Pacific white shrimp, Litopenaeus vannamei

As conserved nuclear factors, Akirins play critical roles in regulating antimicrobial peptides (AMPs) transcription downstream of NF-κB dependent signaling pathways in insects and mammals. However, no any functional studies was reported in penaeid shrimp. The identification and functional analysis of Akirin in the Pacific white shrimp, Litopenaeus vannamei were made in this research. The 833 nucleotides cDNA of Litopenaeus vannamei Akirin (LvAkirin) was obtained with an open reading frame of 639 bp, which encodes a putative protein of 212 amino acids. The molecular weight of LvAkirin is about 23.7 kDa with theoretical pI of 9.05. Two predicted nuclear localization signals (NLSs) were found and amino acid sequence alignments showed that Akirins are highly conserved between insects and mammals. The constitutive expression of LvAkirin mRNA was confirmed in all the examined tissues and high level appeared in testis followed by hemocytes and gill. LvAkirin mRNA was strongly induced in response to Vibrio parahaemolyticus infection. Silencing LvAkirin by dsRNA significantly reduced the expression of NF-κB dependent anti-lipopolysaccharide factor, crustin and penaeidin3a as well as transcription factors, Dorsal and Relish post Vibrio anguillarum (V. anguillarum) and Micrococcus lysodeikticus (M. lysodeikticus) challenge. Antibacterial activities of shrimp plasma was analyzed and high cumulative mortality was found in LvAkirin-silenced shrimps post bacteria challenge. Hence, we proposed LvAkirin might function as a positive nuclear factor of NF-κB dependent signaling pathways in shrimp innate immunity.

p53 is involved in shrimp survival via its regulation roles on MnSOD and GPx in response to acute environmental stresses.

The tumor suppressor gene p53 plays a critical role in safeguarding the integrity of genome in mammalian cells. It acts as a sequence-specific transcription factor. Once activated by a variety of cellular stresses, p53 transactivates downstream target genes, through which it regulates cell cycle and apoptosis. However, little is known about p53 as well as its downstream target genes in invertebrates. A full length cDNA that encodes a 453-amino-acid p53 protein (Lvp53) was characterized in the Pacific white shrimp (Litopenaeus vannamei) to explore the potential relationships between p53 and two antioxidant enzyme genes: Mn-superoxide dismutase (MnSOD) and glutathione peroxidase (GPx) in eliminating cell stresses in L. vannamei. Sequence analysis revealed a close phylogenetic relationship between Lvp53 and that of Marsupenaeus japonicus, and a high degree of conservation in critical amino acids residues is involved in DNA and zinc binding among species. Quantitative real-time PCR showed that Lvp53 was expressed with varied levels in all the 11 tissues under investigation. In response to acute pH challenge, the relative expression of Lvp53 was induced in a pH- and time-dependent manner, with the peak observed at pH 6.1 and after 24 h of treatment, in which condition, both the relative mRNA expressions and the enzymatic activities of LvMnSOD and LvGPx were increased correspondingly. In response to acute cadmium (Cd) exposure, the relative expression of Lvp53 was upregulated in a time- and concentration-dependent manner, with the maximum detected at Cd 6.6 μM and after 48 h of exposure, in which case, both the transcripts and the enzymatic activities of LvMnSOD and LvGPx were also induced. After Lvp53 transcripts were declined by double-strand RNA injection, the relative mRNA expressions of LvMnSOD and LvGPx were decreased correspondingly. Meanwhile, pH 6.1 or 6.6 μM Cd could not induce the transcripts or the enzymatic activities of LvMnSOD or LvGPx any more in Lvp53-silenced shrimp, but increased shrimp mortalities. These results indicated the involvement of Lvp53, LvMnSOD and LvGPx in mediating cell stress caused by suboptimal pH and elevated levels of Cd in L. vannamei, and that the expressions of LvMnSOD and LvGPx were positively regulated by Lvp53, which is a potential mechanism for shrimp to survive the oxidative stress that occurs during short-term exposure to Cd or challenge with acidic pH. This finding will contribute to better understanding of p53 signaling pathways and redox regulation in invertebrate organisms.

Molecular characterization of a p38 MAPK from Litopenaeus vannamei and its expression during the molt cycle and following pathogen infection.

The p38 mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase, has been reported to be involved in innate immunity, development and muscle differentiation. To explore the function of p38 in shrimp, partial cDNA sequence of p38 in Litopenaeus vannamei (designated as Lv-p38) was characterized and the expression of Lv-p38 in hepatopancreas of the shrimp after being infected with Vibrio parahaemolyticus and in muscle of the shrimp at different molt stages was detected by quantitative RT-PCR in this study. The results showed that the open reading frame of Lv-p38 was 1098 bp and encoded a protein of 365 amino acids. The protein of Lv-p38 which showed close phylogenetic relationship to Marsupenaeus japonicus p38 had a conserved TGY motif and serine/threonine protein kinase (S_TKc) domain. The expression of Lv-p38 was detected in all tested tissues, especially in the hepatopancreas and muscle. The expression of Lv-p38 in the hepatopancreas was different from that of the control at the 24th hour after the injection of V. parahaemolyticus and in the muscle significantly increased at stage C but decreased at other stages during molt, illustrating that Lv-p38 could be involved in pathogen infection and the molt cycle of shrimp. In conclusion, we identified Lv-p38 and studied its role in pathogen infection and molting, which might facilitate our understanding of the function of p38 in innate immunity and growth during molt of shrimp.

Molecular characterization of LvAV in response to white spot syndrome virus infection in the Pacific white shrimp (Litopenaeus vannamei)

Litopenaeus vannamei is the most important farmed shrimp species globally, but its production is affected by several factors, including infectious disease. White spot syndrome virus (WSSV), in particular, causes significant shrimp losses. To understand the shrimps immune response against WSSV, we cloned LvAV from L.u2009vannamei and analyzed its expression pattern in different tissues, in addition to its expression following infection. We employed dsRNA and recombinant (r)LvAV to explore the potential role of LvAV in shrimp immunity when infected with WSSV. We find that LvAV is a C-type Lectin composed of 176 amino acids with a signal peptide and a specific C-type Lectin-type domain (CTLD). It shares 81% amino acid similarity with PmAV, an antiviral-like C-type Lectin from Penaeus monodom, and it is highly expressed in the hepatopancreas. Its expression is affected by infection with both WSSV and V.u2009parahaemolyticus. Significantly, injection with rLvAV slowed WSSV replication, while injection with LvAV dsRNA initially led to enhanced virus propagation. Surprisingly, LvAV dsRNA subsequently led to a dramatic decrease in viral load in the later stages of infection, suggesting that LvAV may be subverted by WSSV to enhance viral replication or immune avoidance. Our results indicate that LvAV plays an important, but potentially complex role in the Pacific white shrimps immune defense.

Identification of 10 transcripts of FREP in penaeid shrimp Litopenaeus vannamei.

Fibrinogen-related proteins (FREPs) are widely distributed in vertebrates and invertebrates and known having Fibrinogen-related domains (FReDs) which function in multiple aspects, especially in innate immune response as pattern recognition receptors. However, there is no any report about FREP in penaeid shrimp Litopenaeus vannamei. Here, totally 10 transcripts of FREP were isolated and named LvFREP1.1, 1.2 until 1.10. All of the 10 transcripts have high identity in their 3 ends and encode conserved FReDs. Since the 10 transcripts are highly similar we could not design any primers that can amplify a single transcript. We chose a pair of primers corresponding to part of LvFREP1.1 and LvFREP1.5 to examine their expression. Tissue distribution indicated LvFREP1.1 and LvFREP1.5xa0mRNA locates in hemocytes, gills, intestine, hearts and slightly in nerve. The expression of LvFREP1.1 and LvFREP1.5 behaves differently post bacteria and virus infection. Besides, recombinant LvFReD could agglutinate bacteria Vibrio harveyi in the presence of Ca2+. These initial data presents the diversity of FREPs in penaeid shrimp and also push us to further explore their roles in shrimp immune response.

Identification and characterization of two Croquemort homologues in penaeid shrimp Litopenaeus vannamei

ABSTRACT Croquemort, the homologue of human CD36, is a member of class B scavenger receptors, which is involved in bacteria phagocytosis and cytokins release. However, there is still less information about Croquemort in crustaceans. Here, a Croquemort from Pacific white shrimp Litopenaeus vannamei (LvCroquemort) and its truncated form (LvCroquemort‐S1) cDNA sequences were identified, characterized and their role in bacteria clearance was investigated. The deduced protein of LvCroquemort is 533 amino acids and contains typical domains of CD36: the N‐terminus and C‐terminus in cytoplasm, two transmembrane regions and a large extracellular loop‐like domain. However, LvCroquemort‐S1 losses partial cDNA sequence in its middle and its deduced protein losses the C‐terminal transmembrane region and C‐terminus in cytoplasm, the latter of which is found participating in cytokins release in human CD36. LvCroquemort transcript is highly expressed in gills, hemocytes, testis and slightly in heart, hepatopancreas and nerve. Besides, its responses to bacteria Vibrio anguillarum and white spot syndrome virus were examined. Knock‐down of LvCroquemort by specific dsRNA reduces bacteria clearance. These initial data will help to further understand roles of Croquemort in crustacean innate immunity. HIGHLIGHTSTwo Croquemort homologues were identified and characterized in penaeid shrimp Litopenaeus vannamei.Its responses to bacteria Vibrio anguillarum and white spot syndrome virus were examined.RNAi knock‐down of LvCroquemort reduced bacteria clearance.