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Experiments were performed in triplicate and repeated three times with similar results. Bars display mean+s.d., and statistical analysis was performed using Student’s T test and the P values were provided (**, P<0.01; *, P<0.05).
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Pacific white shrimp (Litopenaeus vannamei), the major species of farmed shrimps in the world, has been attracting extensive studies, which require more and more genome background knowledge. The now available transcriptome data of L. vannamei are insufficient for research requirements, and have not been adequately assembled and annotated.
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Citations
... Based on the L. vannamei transcriptome data in our laboratory (19), a partial cDNA sequence was obtained to amplify the full-length cDNA sequence using the RACE method according to a previously published method (20). In brief, RACE PCR and nested PCR were performed using a SMARTer RACE cDNA amplification kit (Clontech, Dalian, China) in accordance with the manufacturer's instructions. ...
β-Defensins are a family of cysteine-rich antimicrobial peptides that are generally monodomain. Interestingly, the avian β-defensin 11 (AvBD11) is unique, with two β-defensin motifs with a broad range of antimicrobial activities. However, a double-sized β-defensin has not been identified and functionally characterized in invertebrates. In this study, we cloned and identified a double-β-defensin in shrimp Litopenaeus vannamei (named LvDBD) and explored its potential roles during infection with shrimp pathogens Vibrio parahaemolyticus and white spot syndrome virus (WSSV). LvDBD is an atypical double-sized defensin, which is predicted to possess two motifs related to β-defensin and six disulfide bridges. The RNA interference-mediated knockdown of LvDBD in vivo results in phenotypes with increased bacterial loads, rendering the shrimp more susceptible to V. parahaemolyticus infection, which could be rescued by the injection of recombinant LvDBD protein. In vitro, rLvDBD could destroy bacterial membranes and enhance hemocyte phagocytosis, possibly attributable to its affinity to the bacterial wall components LPS and peptidoglycan. In addition, LvDBD could interact with several viral envelope proteins to inhibit WSSV proliferation. Finally, the NF-κB transcription factors (Dorsal and Relish) participated in the regulation of LvDBD expression. Taken together, these results extend the functional understanding of a double-β-defensin to an invertebrate and suggest that LvDBD may be an alternative agent for the prevention and treatment of diseases caused by V. parahaemolyticus and WSSV in shrimp.
... An expressed sequence tag encoding a putative AhR protein was retrieved from L. vannamei transcriptome data analyzed by our laboratory (35). The 39 and 59 ends of LvAhR were obtained with gene-specific primers using the rapid amplification of cDNA ends (RACE) method as previously described (Supplemental Table I) (36). ...
... Gills, hemocytes, and intestines of challenged shrimp were sampled at 0, 4, 8, 12, 24, 36, 48, and 72 h postinjection, and each sample was collected and pooled from 15 shrimp. Total RNA extraction and a quantitative RT-PCR (qRT-PCR) assay were performed according to former descriptions (35). ...
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates immune modulation following exposure of animals to many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood, especially in invertebrates. In this study, a cDNA encoding an AhR homolog was cloned from an arthropod Litopenaeus vannamei (LvAhR). The expression of LvAhR was strongly upregulated in response to the challenge of white spot syndrome virus, a pathogen of highly contagious and fatal infectious disease of shrimp. The relevance of LvAhR to host defense was underlined by heightened susceptibility and elevated virus loads after AhR-silenced shrimp exposure to white spot syndrome virus. LvAhR could induce an apoptosis response through regulating the expression of L. vannamei caspase-1 (homologous to human caspase-3) by directly targeting its promoter that was required to couple with AhR nuclear translocator. Additionally, knockdown of L. vannamei caspase-1 resulted in elevated virus titers and a lower cell apoptotic rate. Thus, we demonstrate that an AhR-caspase axis restrains virus replication by promoting antiviral apoptosis, supporting a previously unidentified direct link between AhR signaling and caspase-mediated apoptosis signaling and, furthermore, suggests that the AhR-caspase axis could be a potential therapeutic target for enhancing antiviral responses in arthropods.
... In Drosophila, the activation of the IMD pathway by bacterial DAPtype PGN from most Gram-negative bacteria and some Gram-positive bacteria requires the sensing receptors of peptidoglycan recognition proteins (PGRPs) [109,122]. Still now, no PGRP homolog is reported and found in shrimps, although we have tried our best to search for homologous sequences in the available transcriptome data from ours [123] and others submitted in NCBI (data not shown). Other sequence unrelated proteins might function as receptors for IMD pathway to sense PGN and initiate signaling in shrimps. ...
... Based on L. vannamei EST and genome data [16], a putative IFI6-16 protein was retrieved to clone the full length of LvIFI6-16 with the gene-specific primers (Table 1) through the rapid amplification cDNA ends (RACE) method. The cDNA library for RACE PCR was constructed with the SMARTer PCR cDNA Synthesis Kit (Clontech) according to the user's instructions. ...
A growing number of evidence shows that some invertebrates possess an antiviral immunity parallel to the interferon (IFN) system of higher vertebrates. For example, the IRF (interferon regulatory factor)–Vago–JAK/STAT regulatory axis in an arthropod, shrimp Litopenaeus vannamei (whiteleg shrimp) is functionally similar to the IRF–IFN–JAK/STAT axis of mammals. IFNs perform their cellular immunity by regulating the expression of target genes collectively referred to as IFN-stimulated genes (ISGs). However, the function of invertebrate ISGs in immune responses is almost completely unclear. In this study, a potential ISG gene homologous to the interferon-induced protein 6-16 (IFI6-16) was cloned and identified from L. vannamei, designated as LvIFI6-16. LvIFI6-16 contained a putative signal peptide in the N-terminal, and a classic IFI6-16-superfamily domain in the C-terminal that showed high conservation to other homologs in various species. The mRNA levels of LvIFI6-16 were significantly upregulated after the stimulation of poly (I:C) and challenges of white spot syndrome virus (WSSV). Moreover, silencing of LvIFI6-16 caused a higher mortality rate and heightened virus loads, suggesting that LvIFI6-16 could play a crucial role in defense against WSSV. Interestingly, we found that the transcription levels of several caspases were regulated by LvIFI6-16; meanwhile, the transcription level of LvIFI6-16 self was regulated by the JAK/STAT cascade, suggesting there could be a JAK/STAT–IFI6-16–caspase regulatory axis in shrimp. Taken together, we identified a crustacean IFI6-16 gene (LvIFI6-16) for the first time, and provided evidence that the IFI6-16 participated in antiviral immunity in shrimp.
... The white shrimp (Litopenaeus vannamei) is a widely cultured crustacean in the Pacific Ocean and is considered one of the dominant shellfish in world aquaculture with a global production of 300 metric tons [1][2][3][4][5][6][7]. The culture of Litopenaeus vannamei (L. ...
... vannamei) is widely extended due its adaptability to different environmental conditions. Currently, several factors could threaten the production of L. vannamei: (1) the culture expansion has a negative impact on the environment, caused by farm effluents, (2) the high density during production has increased disease outbreaks in ponds and (3) emergence of antibiotic resistant pathogens given the continual use of these compounds during the culture [3,4,8,9]. ...
... In this sense, the microorganisms that inhabit the host are capable of producing digestible molecules that improve growth, health status, life-span and the resistance to biotic and abiotic factors in different of shellfish species [2,[9][10][11][12]. The digestive functions in several species of decapods are performed by the hepatopancreas; in this organ the interaction among bacteria and the host allows the absorption of different types of substrates [3,4,10,[12][13][14]. ...
White leg shrimp (Litopenaeus vannamei) is a widely cultured species along the Pacific coast and is one of the most important crustaceans in world aquaculture. The microbiome composition of L. vannamei has been previously studied in different developmental stages, but there is limited information regarding the functional role of the microbiome during the development of L. vannamei. In this study the metatranscriptome in different developmental stages of L. vannamei (larvae, juvenile and adult) were generated using next generation sequencing techniques. The bacterial phyla found throughout all the stages of development belonged to the Proteobacteria, Firmicutes and Actinobacteria, these bacterial phyla are present in the digestive tract and are capable of producing several hydrolytic enzymes, which agrees with high representation of the primary metabolism and energy production, in both host and the microbiome. In this sense, functional changes were observed as the development progressed, in both host and the microbiome, in stages of larvae the most represented metabolic functions were associated with biomass production; while in juvenile and adult stages a higher proportion of metabolic functions associated to biotic and abiotic stress in L. vannamei and the microbiome were shown. This study provides evidence of the interaction of the microbiome with L. vannamei, and how the stage of development and the culture conditions of this species influences the gene expression and the microbiome composition, which suggests a complex metabolic network present throughout the life cycle of L. vannamei.
... Ten Hox genes has been identified in crustaceans, such as in Litopenaeus vannamei (Sun et al., 2015), Penaeus monodon (Uengwetwanit et al., 2021), Daphnia pulex, and Daphnia magna (D. H. Kim et al., 2018;Pace et al., 2016), while nine Hox genes have been identified in Paracyclopina nana (H. S. Kim et al., 2016), Exapalomon carinicauda (Yuan et al., 2017), P. hawaiensis , and Lepas anserifera (Ip et al., 2021). However, the identification and expression of Hox genes during development of crustacean decapods has been limited to P. hawaiensis , Procambarus clarkii (Abzhanov & Kaufman, 2000a, 2000b, L. vannamei (Li et al., 2012;Sun et al., 2015), and Eriocheir sinensis (Cui et al., 2021). ...
Hox genes encode transcription factors that specify the body segment identity during development, including crustaceans, such as amphipods and decapods, that possess a remarkable diversity of segments and specialized appendages. In amphipods, alterations of specialized appendages have been obtained using knockout experiment of Hox genes, which suggests that these genes are involved in the evolution of morphology within crustaceans. However, studies of Hox genes in crustaceans have been limited to a few species. Here, we identified the homeodomain of nine Hox genes: labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abdA), and Abdominal-B (AbdB), and evaluated their expression by RT-qPCR and RT-PCR in the ovary, during embryonic development, and at the first larval stage (Zoea I) of the decapod Macrobrachium olfersii. The transcript levels of lab, Dfd, and ftz decreased and transcripts of pb, Scr, Antp, Ubx, abdA, and AbdB increased during embryonic development. Hox genes were expressed in mature ovaries and Zoea I larval stages, except Scr and ftz, respectively. In addition, isoforms of Dfd, Scr, Ubx, and abdA, which have been scarcely reported in crustaceans, were described. New partial sequences of 87 Hox genes from other crustaceans were identified from the GenBank database. Our results are interesting for future studies to determine the specific function of Hox genes and their isoforms in the freshwater prawn M. olfersii and to contribute to the understanding of the diversity and evolution of body plans and appendages in Crustaceans.
... [8][9][10][11][12] Currently, transcriptomic studies using RNA-Seq, which previously required genomic resources to be developed, can now be applied to species with no previous genomic knowledge, allowing the identification of hundreds of thousands of transcripts of any species of interest. 13 Li et al. 14 were the first to publish the transcriptome of macerated larvae of the P. vannamei shrimp, detecting 73,505 unigenes, with only 37.8% of them found in SwissProt. Using BLAST 15 and BLAST2GO, 16,17 they classified 11,153 unigenes into 25 categories of cluster of orthologous groups, 8171 unigenes were assigned to 51 functional groups of GO and 18,154 unigenes were divided into 220 KEGG pathways. ...
During the last 15 years, a significant amount of data related to the crustacean immune system has been generated, mainly led by the advances made in the field of transcriptomic analysis through massive mRNA sequencing technology. In order to shed new light on the genes and metabolic pathways associated with the immune system in shrimp species, we have carried out a meta‐analysis using the current literature and the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI), which has 1195 published transcriptomic data sets until 31 December 2020, from different tissues and 14 Penaeus species. The transcriptomic analyses published so far have explored the effects of the challenge from viral and bacterial pathogens, as well as abiotic stressors, such as ammonia, temperature, salinity, oxygen deprivation and pH. They have also explored the effect of dietary factors and beneficial microbes (pre‐ and probiotics) in the immune response. In order to harness the vast amount of information available, multidisciplinary efforts have to generate better genomic tools to overcome the complexity of the genome and to implement the knowledge gained into commercial applications for the improvement of the culture conditions, adaptation of more sustainable feeds and producing strains showing more resilience/tolerance to diseases.
... The poor annotation efficiency could be due to insufficient sequences in public databases for phylogenetically closely related species. Some of the unannotated sequences may be untranslated regions, non-coding RNAs, small RNAs, or short sequences which do not contain known protein domains [44]. In addition, 4763 and 619 DEGs were identified in Resting versus Male and Male versus Hermaphrodite group, respectively. ...
Background
Gonad development and differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. However, the mechanisms underlying gonad development and gametogenesis remain unclear in Tridacna squamosa, a large-size bivalve of great ecological value. They are protandrous simultaneous hermaphrodites, with the male gonad maturing first, eventually followed by the female gonads. In this study, nine gonad libraries representing resting, male and hermaphrodite stages in T. squamosa were performed to identify the molecular mechanisms.
Results
Sixteen thousand four hundred ninety-one unigenes were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 5091 and 7328 unigenes were assigned to Gene Ontology categories and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database, respectively. A total of 4763 differentially expressed genes (DEGs) were identified by comparing male to resting gonads, consisting of 3499 which were comparatively upregulated in males and 1264 which were downregulated in males. Six hundred-ninteen DEGs between male and hermaphroditic gonads were identified, with 518 DEGs more strongly expressed in hermaphrodites and 101 more strongly expressed in males. GO (Gene Ontology) and KEGG pathway analyses revealed that various biological functions and processes, including functions related to the endocrine system, oocyte meiosis, carbon metabolism, and the cell cycle, were involved in regulating gonadal development and gametogenesis in T. squamosa. Testis-specific serine/threonine kinases 1 (TSSK1), TSSK4, TSSK5, Doublesex- and mab-3-related transcription factor 1 (DMRT1), SOX, Sperm surface protein 17 (SP17) and other genes were involved in male gonadal development in Tridacna squamosal. Both spermatogenesis- (TSSK4, spermatogenesis-associated protein 17, spermatogenesis-associated protein 8, sperm motility kinase X, SP17) and oogenesis-related genes (zona pellucida protein, Forkhead Box L2, Vitellogenin, Vitellogenin receptor, 5-hydroxytryptamine, 5-hydroxytryptamine receptor) were simultaneously highly expressed in the hermaphroditic gonad to maintain the hermaphroditism of T. squamosa.
Conclusion
All these results from our study will facilitate better understanding of the molecular mechanisms underlying giant clam gonad development and gametogenesis, which can provided a base on obtaining excellent gametes during the seed production process for giant clams.
... The partial cDNA sequence of BigPEN was obtained from transcriptomic sequencing of L. vannamei [24], and its full-length cDNA sequence was cloned by 5 ′ and 3 ′ rapid amplification of cDNA ends (RACE) PCR according to a previously published method [25]. The full-length sequence of PEN2 (accession no. ...
... Penaeidins have been previously identified as AMPs with significant antibacterial and antifungal activities [37]. To explore whether penaeidins have any antiviral roles in the defense against WSSV, we first searched the expressed sequence tag (EST) sequences homologous to known penaeidin proteins from our transcriptome in L. vannamei [24] and obtained a new paralog. We cloned the full-length cDNA sequence of the new paralog by using the rapid amplification cDNA ends (RACE)-PCR method. ...
Emerging studies have indicated that some penaeidins restrict virus infection; however, the mechanism(s) involved are poorly understood. In the present study, we uncovered that penaeidins are a novel family of antiviral effectors against white spot syndrome virus (WSSV), which antagonize the envelope proteins to block viral entry. We found that the expression levels of four identified penaeidins from Litopenaeus vannamei, including BigPEN, PEN2, PEN3, and PEN4, were significantly induced in hemocytes during the early stage of WSSV infection. Knockdown of each penaeidin in vivo via RNA interference resulted in elevated viral loads and rendered shrimp more susceptible to WSSV, while the survival rate was rescued via the injection of recombinant penaeidins. All penaeidins, except PEN4, were shown to interact with several envelope proteins of WSSV, and all four penaeidins were observed to be located on the outer surface of the WSSV virion. Co-incubation of each recombinant penaeidin with WSSV inhibited virion internalization into hemocytes. More importantly, we found that PEN2 competitively bound to the envelope protein VP24 to release it from polymeric immunoglobulin receptor (pIgR), the cellular receptor required for WSSV infection. Moreover, we also demonstrated that BigPEN was able to bind to VP28 of WSSV, which disrupted the interaction between VP28 and Rab7 – the Rab GTPase that contributes to viral entry by binding with VP28. Taken together, our results demonstrated that penaeidins interact with the envelope proteins of WSSV to block multiple viral infection processes, thereby protecting the host against WSSV.
... The hemocytes and intestines of challenged shrimp were sampled at 0, 4, 8, 12, 24, 36, 48, and 72 h post-injection (hpi), and each sample was collected and pooled from 15 shrimp. Total RNA and qRT-PCR were performed as described previously (21). Expression levels of LvTRAF3 were calculated using the Livak (2 − CT ) method after normalization to L. vannamei EF-1a (Accession No. GU136229). ...
Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB- and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069, wsv056, and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway.
