Phenotypic sorting of individual male and female intersex Cherax quadricarinatus and analysis of molecular differences in the gonadal transcriptome.

Cherax quadricarinatus exhibit sexual dimorphism, with males outpacing females in size specification and growth rate. However, there is limited understanding of the molecular mechanisms underlying sex determination and sex differentiation in crustaceans. To study the differences between intersex individuals and normal individuals, this study counted the proportion of intersex individuals in the natural population, collected the proportion of 7 different phenotypes in 200 intersex individuals, and observed the differences in tissue sections. RNA-seq was used to study the different changes in the transcriptome of normal and intersex gonads. The results showed that: the percentage of intersex in the natural population was 1.5 %, and the percentage of different types of intersex ranged from 0.5 % to 22.5 %; the sections revealed that the development of normal ovaries was stagnant at the primary oocyte stage when intersex individuals with ovaries were present; We screened for pathways and genes that may be associated with gonadal development and sex, including ovarian steroid synthesis, estrogen signaling pathway, oocyte meiosis, progesterone-mediated oocyte maturation, etc. Relevant genes including tra2a, dmrta2, ccnb2, foxl2, and smad4. This study provides an important molecular basis for sex determination, sex-controlled breeding, and unisex breeding in red crayfish.

A simple sequence repeats marker of disease resistance in shrimp Litopenaeus vannamei and its application in selective breeding.

The polymorphism of the simple sequence repeat (SSR) in the 5' untranslated coding region (5'-UTR) of the antiviral gene IRF (LvIRF) has been shown to be implicated in the resistance to viral pathogens in shrimp Litopenaeus vannamei (L. vannamei). In this study, we explored the potential of this (CT)n-SSR marker in disease resistance breeding and the hereditary property of disease resistance traits in offspring. From 2018 to 2021, eight populations were generated through crossbreeding by selecting individuals according to microsatellite genotyping. Our results demonstrated that shrimp with the shorter (CT)n repeat exhibited higher resistance to white spot syndrome virus (WSSV) or Decapod iridescent virus 1 (DIV1); meanwhile, these resistance traits could be inherited in offspring. Interestingly, we observed that the longer (CT)n repeats were associated with bacterial resistance traits. Accordingly, shrimp with longer (CT)n repeats exhibited higher tolerance to Vibrio parahaemolyticus infection. Taken together, these results indicate that the single (CT)n-SSR marker could be used to selective breeding for both resistance to virus and bacteria in shrimps.

Signaling events induced by lipopolysaccharide-activated Toll in response to bacterial infection in shrimp.

Toll-like receptors (TLR) play a crucial role in the detection of microbial infections in vertebrates and invertebrates. Mammalian TLRs directly recognize a variety of structurally conserved microbial components. However, invertebrates such as Drosophila indirectly recognize microbial products by binding to the cytokine-like ligand Spätzle, which activates signaling cascades that are not completely understood. In this study, we investigated the signaling events triggered by Toll in response to lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, and Vibrio parahaemolyticus infection in the arthropod shrimp Litopenaeus vannamei. We found that five of the nine Tolls from L. vannamei bound to LPS and the RNAi of LvToll1, LvToll2, LvToll3, LvToll5, and LvToll9 weakened LvDorsal-L phosphorylation induced by V. parahaemolyticus. All nine Tolls combined with MyD88 via the TIR domain, thereby conferring signals to the tumor necrosis factor receptor-associated factor 6 (TRAF6)-transforming growth factor-ß activated kinase 1 binding protein 2 (TAB2)-transforming growth factor-ß activated kinase 1 (TAK1) complex. Further examination revealed that the LvTRAF6-LvTAB2-LvTAK1 complex contributes to Dorsal-L phosphorylation and nuclear translocation during V. parahaemolyticus infection. Overall, shrimp Toll1/2/3/5/9-TRAF6/TAB2/TAK1-Dorsal cascades protect the host from V. parahaemolyticus infection, which provides a better understanding of how the innate immune system recognizes and responds to bacterial infections in invertebrates.

Full-length transcriptome sequences of ridgetail white prawn Exopalaemon carinicauda provide insight into gene expression dynamics during thermal stress.

Marine heat waves and extreme high temperature become more frequent and intense in these years, which affected the survival of aquaculture animals. The ridgetail white prawn Exopalaemon carinicauda is an important economic species in eastern China, which has remarkable thermal tolerance. However, there has been little study of its thermal-adaptation mechanisms due to the complex genetic structure and unknown genome. To better understand the molecular mechanisms of E. carinicauda to adapt to the changing temperature, a combination of Illumina-based short reads RNA-seq and single molecule real-time-based full-length transcriptome sequencing was used in this study. In total, 17,212 unigenes from high-quality transcripts of E. carinicauda were generated and 14,663 complete ORFs were detected with an average length of 1980 bp. In addition, the transcriptome profiles of E. carinicauda treated with 34 °C heat stress for 6 and 24 h were analyzed. These differentially expressed genes were primarily enriched in oxidation-reduction process (Gene Ontology enrichment, GO) and the pathways of starch and sucrose metabolism (Kyoto Encyclopedia of Genes and Genomes enrichment, KEGG) after 6 h thermal stress, which indicated that E. carinicauda was suffering the attack by reactive oxygen species. After 24 h thermal stress, these differentially expressed genes were enriched in the pathway of lysosome, glycine, serine and threonine metabolism, fatty acid metabolism (KEGG), which indicated the oxidative stress was decreased. Interestingly, 40 genes for hemocyanin were found to be downregulated after 6 h heat stress, which indicated that the immunocompetence of E. carinicauda decreased after short term thermal stress (6 h). After 24 h thermal stress, E. carinicauda showed transcriptional adaptation to high temperature by upregulating of 11 genes encoding molecular chaperones, including HSP40 and HSP90 which were firstly reported to be related to thermal stress in E. carinicauda. These results promote a better understanding of the thermal-adaptation mechanism of E. carinicauda.

Shrimp TAB1 interacts with TAK1 and p38 and activates the host innate immune response to bacterial infection.

Mammalian TAB1 has been previously identified as transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) binding protein, which functions as the activator of TAK1 and p38. This report, for the first time, identified and characterized the homolog of TAB1 in shrimp, to be specific, the homolog gene from Litopenaeus vannamei, containing a 1560-bp open reading frame (ORF) that encoded a putative protein of 519 amino acids with the conserved PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain in N-terminal and a TAK1 binding motif in C-terminus, has been cloned and named LvTAB1. LvTAB1 was most abundant in gills and its expression could respond significantly to a series of stimuli, including LPS, Vibrio parahemolyticus and Staphylococcus aureus. Moreover, Co-immunoprecipitation (Co-IP) experiments showed that LvTAB1 could combine with LvTAK1 as well as Lvp38, two members of IMD-NF-κB/MAPK pathway, which meant LvTAB1 could have a role in regulating the activities of these kinases. Over-expression of LvTAB1 in drosophila S2 cells could improve the transcriptional levels of antimicrobial peptide genes (AMPs) such as Diptericin (Dpt), the hallmark of drosophila NF-κB activated genes, indicating its activation effect on NF-κB pathway. Furthermore, suppression of LvTAB1 expression in vivo by RNA-interference increased the sensibility of shrimps to V. parahaemolyticus infection, implying its protective role against bacterial infection. In conclusion, these results provide some insight into the function of LvTAB1 during bacterial infection.

Identification of two p53 isoforms from Litopenaeus vannamei and their interaction with NF-κB to induce distinct immune response.

p53 is a transcription factor with capability of regulating diverse NF-κB dependent biological progresses such as inflammation and host defense, but the actual mechanism remains unrevealed. Herein, we firstly identified two novel alternatively spliced isoforms of p53 from Litopenaeus vannamei (LvΔNp53 and the full-length of p53, LvFLp53). We then established that the two p53 isoforms exerted opposite effects on regulating NF-κB induced antimicrobial peptides (AMPs) and white spot syndrome virus (WSSV) immediate-early (IE) genes expression, suggesting there could be a crosstalk between p53 and NF-κB pathways. Of note, both of the two p53 isoforms could interact directly with LvDorsal, a shrimp homolog of NF-κB. In addition, the activation of NF-κB mediated by LvDorsal was provoked by LvΔNp53 but suppressed by LvFLp53, and the increased NF-κB activity conferred by LvΔNp53 can be attenuated by LvFLp53. Furthermore, silencing of LvFLp53 in shrimp caused higher mortalities and virus loads under WSSV infection, whereas LvΔNp53-knockdown shrimps exhibited an opposed RNAi phenotype. Taken together, these findings present here provided some novel insight into different roles of shrimp p53 isoforms in immune response, and some information for us to understand the regulatory crosstalk between p53 pathway and NF-κB pathway in invertebrates.

An invertebrate STING from shrimp activates an innate immune defense against bacterial infection.

It has been proposed that invertebrate stimulators of interferon genes (STINGs) do not take part in the innate immune response to infection. Herein, we identified a new STING homolog from pacific white shrimp Litopenaeus vannamei (LvSTING). Some amino acids crucial for recognizing cyclic dinucleotides in mammals are highly conserved in LvSTING. Moreover, LvSTING expression can be robustly induced by challenge with the Gram-negative bacteria Vibrio parahaemolyticus. Silencing of LvSTING contributes to decreased expression of the antimicrobial peptide PEN4 and renders shrimp more susceptible to V. parahaemolyticus infection, while coinjection with the recombinant LvSTING protein can rescue PEN4 expression in vivo and confer shrimp with more resistance to infection. Taken together, these results suggest that LvSTING is involved in the innate immune response to bacterial infection.

Identification and functional analysis of a TEP gene from a crustacean reveals its transcriptional regulation mediated by NF-κB and JNK pathways and its broad protective roles against multiple pathogens.

Thioester-containing proteins (TEPs) are present in a wide range of species from deuterostomes to protostomes and are thought to be involved in innate immunity. In the current study, a TEP gene homologous to insect TEPs (iTEP) from the crustacean Litopenaeus vannamei, named LvTEP1, is cloned and functionally characterized. The open reading frame (ORF) of LvTEP1 is 4383 bp in length, encoding a polypeptide of 1460 amino acids with a calculated molecular weight of 161.1 kDa LvTEP1, which is most similar to other TEPs from insects, contains some conserved sequence features, including a N-terminal signal peptide, a canonical thioester (TE) motif, and a C-terminal distinctive cysteine signature. LvTEP1 is expressed in most immune-related tissues, such as intestine, epithelium, and hemocytes, and the mRNA level of LvTEP1 is upregulated in hemocytes after bacterial and viral challenges, indicating its involvement in the shrimp innate immune response. An expression assay in Drosophila S2 cells shows LvTEP1 to be a full-length secretory protein, and processed forms are present in the supernatant. Of note, only the processed form of LvTEP1 protein can bind to both the gram-negative bacterium Vibrio parahaemolyticus and the gram-positive bacterium Staphylococcus aureus in vitro, and its abundance can be induced after bacterial treatment. Moreover, knockdown of LvTEP1 renders shrimps more susceptible to both V. parahaemolyticus and S. aureus, as well as white spot syndrome virus (WSSV) infection, suggesting its essential defensive role against these invading microbes. We also observe that the expression of LvTEP1 is regulated in a manner dependent on both NF-κB and AP-1 transcription factors in naive shrimps and in vitro, suggesting that LvTEP1 could be poised in the body cavity prior to infection and thus play an important role in basal immunity. Taken together, our findings provide some in vitro and in vivo evidence for the involvement of LvTEP1 in shrimp innate immunity and provide some insight into its expression regulation mediated by multiple transcription factors or signaling pathways.

A MicroRNA-Mediated Positive Feedback Regulatory Loop of the NF-κB Pathway in Litopenaeus vannamei.

In the evolutionarily conserved canonical NF-κB pathway, degradation of the NF-κB inhibitor IκB in the cytoplasmic NF-κB/IκB complex allows the liberated NF-κB to translocate into the nucleus to activate various target genes. The regulatory mechanism governing this process needs further investigation. In this study, a novel microRNA, temporarily named miR-1959, was first identified from an invertebrate Litopenaeus vannamei miR-1959 targets the 3'-untranslated region of the IκB homolog Cactus gene and reduces the protein level of Cactus in vivo, whereas the NF-κB homolog Dorsal directly binds the miR-1959 promoter to activate its transcription. Therefore, miR-1959 mediates a positive feedback regulatory loop, in that Dorsal activates miR-1959 expression, and in turn, miR-1959 inhibits the expression of Cactus, further leading to enhanced activation of Dorsal. Moreover, miR-1959 regulates the expression of many antimicrobial peptides in vivo and is involved in antibacterial immunity. To our knowledge, it is the first discovery of a microRNA-mediated feedback loop that directly regulates the NF-κB/IκB complex. This positive feedback loop could collaborate with the known NF-κB/IκB negative loop to generate a dynamic balance to regulate the activity of NF-κB, thus constituting an effective regulatory mechanism at the critical node of the NF-κB pathway.

Presence of Tube isoforms in Litopenaeus vannamei suggests various regulatory patterns of signal transduction in invertebrate NF-κB pathway.

The toll-like receptor (TLR)/NF-κB signaling pathways play critical roles in the innate immune system. The intracellular signal transduction of most TLR pathways in invertebrate cells is triggered by formation of a heterotrimeric complex composed of MyD88, Tube and Pelle. In this study, we identified a Litopenaeus vannamei Pelle (LvPelle) and an isoform of L. vannamei Tube (LvTube) designated as LvTube-1. The interactions among LvPelle, LvTube/LvTube-1 and LvMyD88/LvMyD88-1 were elucidated and their functions during pathogen infections were investigated. Knockdowns of LvPelle and LvTube/LvTube-1 using RNAi strategy led to higher mortalities of shrimps during Vibrio parahemolyticus infection, and could reduce the genome copy number of white spot syndrome virus (WSSV) in the infected muscle tissue but did not affect the mortality caused by WSSV infection. The effects of LvPelle and LvTube/LvTube-1 on promoters containing NF-κB binding motifs were analyzed by dual-luciferase reporter assays and the results demonstrated that LvTube-1 could activate the NF-κB activity to significantly higher level than LvTube did. Moreover, tissue distributions of LvTube and LvTube-1 mRNAs and their expression profiles during pathogen and immune stimulant challenges were different, indicating that they could play different roles in immune responses. This is the first report of Tube isoforms in invertebrates. Together with our previous study on LvMyD88 isoforms, our results suggest that various isoforms of adaptor components may be involved in various regulatory patterns of signal transduction in invertebrate TLR/NF-κB pathway and this could be a strategy adopted by invertebrates to modulate immune responses.

Identification and functional study of a shrimp Dorsal homologue.

Rel/NF-kappaB transcription factors play central roles in induction and regulation of innate immune responses. Here, identification and functional analysis of LvDorsal, a Dorsal homologue from the Pacific white shrimp Litopenaeus vannamei, were described. The full-length cDNA of LvDorsal is 2204bp with an open reading frame that encodes 400 amino acids. The deduced LvDorsal contains a conserved Rel homology domain (RHD), an IPT (Ig-like, plexins and transcription factors) domain and a nucleus localization signal, suggesting that it belongs to the class II NF-kappaB. RT-PCR analysis showed that LvDorsal mRNAs were expressed in all the tissues tested, including gill, epidermis, hemocytes, intestine, stomach, eyestalk, brain, hepatopancreas, muscle, heart and pyloric caecum. Immunofluorescence assay showed that recombinant LvDorsal was translocated into the nucleus of Drosophila S2 cells. Electrophoretic mobility shift assay illustrated that recombinant LvDorsal RHD from S2 cells bound specifically with D. melanogaster kappaB motifs. Additionally, the dual-luciferase reporter assays indicated that LvDorsal could transactivate the reporter gene controlled by the 5' flanking region of shrimp penaeidin-4 and Drosophila attacin genes, suggesting that LvDorsal can regulate the transcription of shrimp penaeidin-4 gene. Study of LvDorsal will help us to better understand shrimp immunity and may help to obtain more effective methods to prevent shrimp diseases.

A novel prophenoloxidase 2 exists in shrimp hemocytes.

The prophenoloxidase (proPO)-activating system in crustaceans and other arthropods is regarded as a constituent of the immune system and plays an important role in defense against pathogens. Hitherto in crustaceans, only one proPO gene per species has been identified. Here we report the identification of a novel proPO-2 (LvproPO-2) from the hemocytes of Litopenaeus vannamei, which shows 72% identity to proPO-1 (LvproPO-1) cloned previously. Northern blotting analysis and quantitative real-time PCR reveal that LvproPO-2 is mainly expressed in the hemocytes, and its expression is down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Western blotting analysis shows that most LvproPO-2/LvPO-2 (L. vannamei phenoloxidase-2) exists in the hemocytes, but not in plasma of L. vannamei. LvproPO-2/LvPO-2 could be detected on the hemocyte surface and the nucleus of hemocytes by indirect immunofluorescence assay (IFA). These findings provide insight into the molecular biological basis for further studying on the defense mechanism of shrimp innate immunity, especially on the proPO-activating system and melanization cascade of shrimp.

A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity.

C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type carbohydrate recognition domain with an EPN (Glu(99)-Pro(100)-Asn(101)) motif that has a predicted ligand binding specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically expressed in the hepatopancreas of L. vannamei. Recombinant LvCTL1 (rLvCTL1) had hemagglutinating activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1 to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that has direct anti-WSSV activity.

Changes in mortality and immunological variables of Litopenaeus vannamei parents and their filial families infected with white spot syndrome under different experimental conditions.

In order to find changes in mortality and immunological variables of Litopenaeus vannamei parents and the filial WSSV-resistant and -susceptible families after infection with WSSV under different experimental conditions, the haemolymph total haemocyte count (THC), phenoloxidase (PO), and superoxide dismutase (SOD) activities were measured at days 0, 1, 3, 6, 9, 12 and 15 after challenge and shrimp mortality was also recorded. When shrimps were challenged with 10(-3) (1.29x10(6)copiesmL(-1)), 10(-4) (1.29x10(5)copiesmL(-1)) or 10(-5) (1.29x10(4)copiesmL(-1)) WSSV stock solution (0.1mLshrimp(-1)), the cumulative mortalities (mean+/-S.E.) on day 15 were 100+/-0%, 79.3+/-1.1%, and 21.7+/-2.3%, respectively. Among shrimps challenged with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)), the cumulative mortalities (mean+/-S.E.) on day 15 in high-density (100shrimpsm(-3)), middle-density (50shrimpsm(-3)), and low-density (25shrimpm(-3)) groups were 95.5+/-0%, 84.7+/-0%, and 72.3+/-0%, respectively. The immunological variables including THC, PO, and SOD were decreased significantly at the beginning of infection stage, while these immunological variables for survivors reached almost the similar levels to the non-infection control group on day 15 after challenge with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)). Cumulative mortality (mean+/-S.E.) on day 15 in 17 filial families (G(2)) ranged from 13.3+/-1.9% to 100+/-0% when shrimps were challenged with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)). Although, the PO and SOD activities for shrimps in the WSSV-resistant family were slightly higher than those in the WSSV-susceptible family at the same sampling time after infection, these differences were not significant (p<0.05).

Characterization of a prophenoloxidase from hemocytes of the shrimp Litopenaeus vannamei that is down-regulated by white spot syndrome virus.

Previously, a prophenoloxidase (proPO) gene (named proPO-a here) from hemocytes of Litopenaeus vannamei was isolated. Here, a proPO-b gene was also identified and characterized from hemocytes of L. vannamei. The cDNA sequences of proPO-a and proPO-b were compared, and it was found that both proPOs had a microsatellite DNA site near the 3' end of the open reading frame (ORF). However, the microsatellite DNA of proPO-b contained a compound imperfect simple sequence repeats (SSR) ((CT)(38)(CA)(8)(AA)(CA)(3)(TA)(CA)(14)), which was different from the perfect one ((CT)(20)) of proPO-a, and the cDNA sequences of proPO-a and proPO-b prior to the microsatellite DNA were almost identical, but differed after the microsatellite DNA. ProPO-b (3232 bp) was longer than proPO-a (2471 bp). The 3' UTR sequence after SSR of proPO-a was not detected in shrimp randomly collected from five different geographically separate populations by reverse-transcription polymerase chain reaction (RT-PCR). On the contrary, the 3' UTR sequence of proPO-b was detected in all five groups of shrimps. Northern blot analysis showed that a transcript at approximately 3.2kb, but not 2.5kb, was detected mainly in hemocytes, and also present in midgut, gill, heart, stomach, posterior midgut cecum, and cuticular epidermis, but no signal was detected in hepatopancreas and musculature. RT-PCR and quantitative real-time RT-PCR analysis showed similar results of the proPO-b expression profile in these shrimp tissues. We also observed that proPO-b expression was down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Our results suggest that proPO-b is a main transcript form of proPO gene in L. vannamei, and it may play a role in defence against WSSV virus.

Profiling of differentially expressed genes in hepatopancreas of white spot syndrome virus-resistant shrimp (Litopenaeus vannamei) by suppression subtractive hybridisation.

In order to find immune-relevant factors responsible for virus resistance and response to the virus infection, the suppression subtractive hybridisation method was employed to identify differentially expressed genes and their expression profiles in the hepatopancreas of the white spot syndrome virus (WSSV) resistant and susceptible Pacific white shrimp (Litopenaeus vannamei). Two forward subtractive libraries (at 0 and 48h time point) and two reverse subtractive libraries (at 0 and 48h time point) were constructed, and more than 1200 clones were sequenced, of which 40 differentially expressed genes were identified. These genes encode proteins corresponding to a wide range of functions, including defence-related proteins, enzymes, transcription factors, apoptotic-related proteins, intracellular components potentially related to signaling cascades, metabolic proteins, and cytoskeletal protein. Five genes (laccase, carboxypeptidase B, H(+)-transporting ATP synthase, Acyl-ConA-binding protein (ACBP), and cortical granule protein with LDL-receptor) are found for the first time in shrimp and their expressions were up-regulated in the virus-resistant shrimp. Among the 40 genes, 30 showed up-regulation in the virus-resistant shrimp comparing with susceptible shrimp, while 10 genes showed down-regulation. Haemocyanin was the most abundant gene in our forward subtractive libraries. In addition, chathepsin L, ecdysteroid regulated protein, zinc proteinase, lectin, sterol carrier protein-X, lysozyme, cortical granule protein with LDL-receptor, leucine-rich repeat LGI family, fatty acid binding protein, and preamylase all showed up-regulation in the resistant shrimp. Furthermore, a number of genes encoding apoptotic-related proteins and antioxidant enzymes were expressed at a higher level in the virus-resistant shrimp. The high expression of the immune-relevant genes in response to the virus infection provides a new insight for further study in the shrimp innate immunity.