Transcriptome analysis of the unfolded protein response in hemocytes of Litopenaeus vannamei.

In this study, Litopenaeus vannamei was injected with double-stranded RNA (dsRNA) against L. vannamei immunoglobulin heavy chain binding protein (LvBip) to activating UPR in the hemocytes, shirmps injected dsRNA against enhanced green fluorescence protein (eGFP) as control group. And genes expression in hemocytes of then were analyzed using Illumina Hiseq 2500 (PE100). By comparing the analyzed results, 1418 unigenes were significantly upregulated, and 596 unigenes were significantly down-regulated upon UPR. Analysis of the differentially expressed genes against known databases indicated that the distribution of gene pathways between the upregulated and down-regulated genes were substantially different. A total of 208 genes of UPR system were obtained, and 69 of them were differentially expressed between the two groups. Results also showed that L. vannamei UPR was involved in various metabolic processes, such as glycometabolism, lipid metabolism, amino acid metabolism, and nucleic acid metabolism. In addition, UPR was emgaged in immune-assicoated signaling pathways, such as NF-κB signaling pathway, NOD-like receptor signaling pathway, Hippo signaling pathway, p38 MAPK signaling pathway and Wnt signaling pathway in L. vannamei. These results improved our current understanding of the L. vannamei UPR, and highlighted its importance in cell homeostasis upon environmental stress.

Identification and characterization of a mitochondrial unfolded protein response transcription factor ATFS-1 in Litopenaeus vannamei.

A mitochondrial specific stress response termed mitochondrial unfolded protein response (UPR(mt)) is activated in responding to disturbance of protein homeostasis in mitochondria. The activating transcription factor associated with stress-1 (designated as ATFS-1) is the key regulator of UPR(mt). To investigating the roles of ATFS-1 (LvATFS-1) in Litopenaeus vannamei mitochondrial stress remission and immunity, it's full length cDNA was cloned. The open reading frame of LvATFS-1 was 1, 557 bp in length, deducing to a 268 amino acids protein. LvATFS-1 was highly expressed in muscle, hemocytes and eyestalk. Subcellular location assays showed that N-terminal of LvATFS-1 contained a mitochondrial targeting sequence, which could directed the fused EGFP located to mitochondria. And the C-terminal of LvATFS-1, which had a nuclear localization signal, expressed in nucleus. The in vitro experiments verified that LvATFS-1 could reduced the level of intracellular reactive oxygen species (ROS). And results of real-time RT-PCR indicated that LvATFS-1 might scavenge excess ROS via ROS-eliminating genes regulation. Reporter gene assays showed that LvATFS-1 could upregulated the expression of the antimicrobial peptide genes in Drosophila Schneider 2 cells. Results of real-time RT-PCR showed that Vibrio alginolyticus or white spot syndrome virus (WSSV) infection induced the expression of LvATFS-1. And knocked-down LvATFS-1 by RNAi resulted in a higher cumulative mortality of L. vannamei upon V. alginolyticus or WSSV infection. These results suggested that LvATFS-1 not only rolled in mitochondrial specific stress responding, but also important for L. vannamei immunologic defence.

Down-regulation apoptosis signal-regulating kinase 1 gene reduced the Litopenaeus vannamei hemocyte apoptosis in WSSV infection.

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is crucial in various cellular responses. In the present study, we identified and characterized an ASK1 homolog from Litopenaeus vannamei (LvASK1). The full-length cDNA of LvASK1 was 5400 bp long, with an open reading frame encoding a putative 1420 amino acid protein. LvASK1 was highly expressed in muscle, hemocyte, eyestalk and heart. Real-time RT-PCR analysis showed that the expression of the LvASK1 was upregulated during the white spot syndrome virus (WSSV) challenge. The knocked-down expression of LvASK1 by RNA interference significantly reduced the apoptotic ratio of the hemocytes collected from WSSV-infected L. vannamei. Furthermore, the down-regulation of LvASK1 also decreased the cumulative mortality of WSSV-infected L. vannamei. These results suggested that down-regulation of LvASK1 decreased the apoptotic rate of hemocytes in WSSV-infected shrimp, and that it could contribute to the reduction of cumulative mortality in WSSV-infected L. vannamei.

Flightless-I (FliI) is a potential negative regulator of the Toll pathway in Litopenaeus vannamei.

Flightless-I (FliI) is a protein negatively modulates the Toll-like receptor (TLR) pathway through interacting with Myeloid differentiation factor 88 (MyD88). To investigate the function of FliI in innate immune responses in invertebrates, Litopenaeus vannamei FliI (LvFliI) was identified and characterized. The full-length cDNA of LvFliI is 4, 304 bp long, with an open reading frame (ORF) encoding a putative protein of 1292 amino acids, including 12 leucine-rich repeat (LRR) domains at the N-terminus and 6 gelsolin homology (GEL) domains at the C-terminus. The LvFliI protein was located in the cytoplasm and LvFliI mRNA was constitutively expressed in healthy L. vannamei, with the highest expression level in the muscle. LvFliI could be up-regulated in hemocytes after lipopolysaccharide (LPS), poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV) challenges, suggesting a stimulation response of LvFliI to bacterial and immune stimulant challenges. Upon LPS stimulation, overexpression of LvFliI in Drosophila Schneider 2 cells led to downregulation of Drosophila and shrimp antimicrobial peptide (AMP) genes. Knockdown of LvFliI by RNA interference (RNAi) resulted in an increase of the expression of three shrimp AMP genes (PEN2, crustin, and Lyz1). However, the mortality rates of LvFliI-knockdown shrimp in response to V. parahaemolyticus, S. aureus or WSSV infections were not significantly different from those of the control group. Taken together, all the results suggested that LvFliI may play a negative role in TLR signaling response in L. vannamei.

Identification and functional characterization of heat shock transcription factor1 in Litopenaeus vannamei.

Heat shock transcription factors belong to the heat shock factor (HSF) protein family, which are involved in heat shock protein (HSP) gene regulation. They are critical for cell survival upon exposure to harmful conditions. In this study, we identified and characterized a HSF1 (LvHSF1) gene in Litopenaeus vannamei, with a full-length cDNA of 2841 bp and an open reading frame encoding a putative protein of 632 amino acids. Through multiple sequence alignment and phylogenetic analysis, it was revealed that LvHSF1 was closed to insect HSF family, which contained a highly conserved DNA-binding domain, oligomerization domains with HR-A/B, and a nuclear localization signal. Tissues distribution showed that LvHSF1 was widely expressed in all tissues tested. And it was upregulated in hemocytes and gills after Vibrio alginolyticus or Staphylococcus aureus infection. Dual-luciferase reporter assays indicated that LvHSF1 activated the promoters of L. vannamei HSP70 (LvHSP70) and L. vannamei Cactus (LvCactus), while inhibited the expressions of Drosophila antimicrobial peptide (AMP) Atta, Mtk, and L. vannamei AMP PEN4 through NF-κB signal transduction pathway modification. Knocked-down expression of LvHSF1 by dsRNA resulted in downregulations of LvHSP70 and LvCactus, as well as cumulative mortality decreasing under V. alginolyticus or S. aureus infection in L. vannamei. Taken together, our data strongly suggest that LvHSF1 is involved in LvHSP70 regulation, therefore plays a great role in stress resistance. And it also takes part in LvCactus/LvDorsal feedback regulatory pathway modification of L. vannamei, which is in favor of V. alginolyticus or S. aureus infection.

An activating transcription factor of Litopenaeus vannamei involved in WSSV genes Wsv059 and Wsv166 regulation.

Members of activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element binding protein (ATF/CREB) family are induced by various stress signals and function as effector molecules. Consequently, cellular changes occur in response to discrete sets of instructions. In this work, we found an ATF transcription factor in Litopenaeus vannamei designated as LvATFß. The full-length cDNA of LvATFß was 1388 bp long with an open reading frame of 939 bp that encoded a putative 313 amino acid protein. The protein contained a basic region-leucine zipper (bZip) domain that was a common feature among ATF/CREB transcription factors. LvATFß was highly expressed in intestines, gills, and heart. LvATFß expression was dramatically upregulated by white spot syndrome virus (WSSV) infection. Pull-down assay revealed that LvATFß had strong affinity to promoters of WSSV genes, namely, wsv059 and wsv166. Dual-luciferase reporter assay showed that LvATFß could upregulate the expression of wsv059 and wsv166. Knocked down LvATFß resulted in decreased expression of wsv059 and wsv166 in WSSV-challenged L. vannamei. Knocked down expression of wsv059 and wsv166 by RNA interference inhibited the replication and reduce the mortality of L. vannamei during WSSV challenge inoculation. The copy numbers of WSSV in wsv059 and wsv166 knocked down group were significant lower than in the control. These results suggested that LvATFß may be involved in WSSV replication by regulating the expression of wsv059 and wsv166.

Mechanical properties of municipal solid waste under different stress paths: Effects of plastic content and particle gradation.

Plastics within municipal solid waste (MSW) are non-degradable. As MSW continues to degrade, the relative content of plastics rises, and particle gradation may also change. Moreover, throughout the landfilling process, MSW is subjected to various stress conditions, potentially influencing its mechanical properties. This study explored the effects of varying plastic contents, different particle gradations, and distinct stress paths on the mechanical properties of MSW, and consolidated drained triaxial tests of 42 groups of reconstituted MSW specimens were conducted. The results showed that there was an optimal plastic content of 6-9 % for MSW, where the shear strength of MSW was higher than that of MSW with other plastic contents. When the stress path changed from TC45 to TC72, the optimal plastic content of MSW changed from 6 % to 9 %. As the plastic content increased, both the cohesion and internal friction angle of the MSW initially increased, then subsequently decreased. The impact of plastic content on cohesion was more pronounced than on the internal friction angle, especially at larger strains. Under various stress paths, MSW with distinct particle size distributions demonstrated diverse stress-strain behaviors. Traditional criteria for evaluating well-graded conditions in soils are not suitable for MSW. The effect of gradation on the cohesion of MSW is essentially due to the predominant role of fiber content; the relationship between gradation and the internal friction angle in MSW is complex and correlates closely with the content of both coarse and fine particles, as well as fibers. This study serves as an essential reference for predicting deformations in landfills and analyzing the stability of landfill slopes.

Molecular characterization and function of a p38 MAPK gene from Litopenaeus vannamei.

p38 mitogen-activated protein kinases (MAPKs) are broadly expressed from yeasts to mammals, and are involved in the regulation of cells responsible to various extracellular stimuli. In this study, a p38 MAPK gene (designated as Lvp38) from Litopenaeus vannamei, was cloned and characterized. It contained the conserved structures of a Thr-Gly-Tyr (TGY) motif and a substrate-binding site, Ala-Thr-Arg-Trp (ATRW). The tissue distribution patterns showed that Lvp38 was widely expressed in all examined tissues, with the highest expression in hemocytes, nerves, and intestines. Quantitative real-time PCR revealed that Lvp38 was upregulated in gills and hemocytes after infection with the Gram-negative Vibrio alginolyticus and the Gram-positive Staphylococcus aureus. Reporter gene assays indicated that Lvp38 activated the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp. Knockdown of Lvp38 by RNA interference (RNAi) resulted in a higher mortality of L. vannamei under V. alginolyticus and S. aureus infection, as well as a reduction in the expression of three shrimp AMP genes, namely, PEN4, crustin, and ALF2. Taken together, our data indicated that Lvp38 played a role in defending against bacterial infections.

Machine learning-based models for predicting gas breakthrough pressure of porous media with low/ultra-low permeability.

Gas breakthrough pressure is a significant parameter for the gas exploration and safety evaluation of engineering barrier systems in the carbon dioxide storage, remediation of contaminated sites, and deep geological repository for disposal of high-level nuclear waste, etc. Test for determining gas breakthrough pressure is very difficult and time-consuming, due to the low/ultra-low conductivity of the specimen. It is also difficult to get a comprehensive and high-precision model based on limited results obtained through individual experiments, as the measurements of gas breakthrough pressure were influenced by many factors. In this study, a collected database was built that covered a lot of former test data, and then, two models were developed by the random forest (RF) algorithm and multiexpression programming (MEP) method. The MEP model constructed with explicit expressions for the gas breakthrough pressure overcame the drawbacks of common "black box" models. Meanwhile, five significant indicators were selected from ten common features using the permutation importance algorithm. The RF model was interpreted by the Shapley value and the PDP/ICE plots, while the MEP model was analyzed through the proposed explicit expression, showing strong consistence with that in former studies. Finally, robustness analysis was conducted, and stability of the proposed two models was verified.

Laboratory investigation on the retention performance of a soil-bentonite mixture used as an engineered barrier: insight into the effects of ionic strength and associated heavy metal ions.

Soil-bentonite (S-B) materials are promising backfill materials for use as engineered barriers in heavy metal-contaminated sites. The effects of contaminant exposure on the retention performance of the S-B barrier remain unrevealed. In this study, based on the pollution status of an abandoned ferroalloy factory located in southern China, the retention performance of the S-B mixture toward Cr(VI) and Zn(II) was studied through adsorption and diffusion experiments sequentially; the separate effect of ionic strength (binary solution) and the combined effect of ionic strength and associated heavy metal ion (ternary solution) were discussed. In NaCl-Cr(VI)/Zn(II) binary solutions, the adsorption of Zn(II) onto the S-B mixture is larger than that of Cr(VI). Kd, Qmax, and ɛacc (accessible porosity) of Cr(VI) increase through increasing ionic strength, while Zn(II) shows the opposite trend; De (effective diffusion coefficient) values for both Cr(VI) and Zn(II) increased with increasing ionic strength and follow a sequence of Cr(VI) > Zn(II), indicating a better retention performance of the S-B mixture to Zn(II). For a given ionic strength, the adsorption of Zn(II) was larger than that of Cr(VI), which can be attributed to the retention specificity of the S-B mixture to anion and cation. In Cr(VI)-Zn(II)-NaCl ternary solutions, the adsorptions of Cr(VI) and Zn(II) are enhanced in varying degrees when compared with their binary solution, which probably could be attributed to the ion bridge role of Cr(VI)/Zn(II) to connect each other that relatively increased the adsorption capacity of S-B material. This work will contribute to an in-depth understanding of the retention performance of the S-B mixture in complicated chemical environments and facilitate the selection of future remediation strategies.

Spatiotemporal distribution and pollution control of pollutants in a Cr(VI)-contaminated site located in Southern China.

Soil and groundwater Cr(VI) pollution resulting from improper disposal and accidental spills is a critical problem worldwide. In this study, a comprehensive study was conducted to assess the hydrogeological conditions of a contaminated site, obtain spatiotemporal distribution and trend forecasts of pollutant Cr(VI), and determine the feasibility of applying clayey engineered barriers for pollution control. The results showed that the hydraulic conductivity (K) of the clayey barrier (1.56E-5 m/d) is several orders of magnitude lower than that of the stratum beneath the contaminated site, with K values ranging from 0.0014 to 4.76 m/d. Cr(VI) exhibits high mobility and a much higher concentration in the vadose zone, with maximum values of 6100 mg/kg in topsoil and 2090 mg/L in the perched aquifer. The simulation results indicated that the groundwater in the vicinity of the contaminated site, as well as downstream of the Lianshui River, is seriously threatened by Cr(VI). Notably, the pollution plume could occur downstream of the Lianshui River after 8 years. The retention efficiency of clayey engineered barriers will decrease over time, at 61.6% after 8 years and 33% after 20 years. This work contributes to an in-depth understanding of Cr(VI) migration at contaminated sites.

The sorption performance of corroded Gaomiaozi bentonite by evolved cement water at different temperatures: the case of europium removal.

In the Chinese high-level radioactive waste geological disposal program, Gaomiaozi (GMZ) bentonite has been selected as the potential buffer/backfill material. After the closure of the repository, the Ca-OH-type alkaline solution (evolved cement water) released by cement degradation may last for more than 100,000 years. The bentonite will undergo the corrosion of evolved cement water (ECW) for a long period. This work focuses on the sorption property of GMZ bentonite altered by ECW. Firstly, the corrosion experiments on compacted GMZ specimens with the dry density of 1.70 Mg/m3 were carried out under constant volume conditions at two temperatures. Then, the sorption of europium (Eu (III)) onto the corroded GMZ bentonite was studied by batch experiments. The results of batch sorption tests indicate that the altered GMZ bentonite keeps an effective removal property with the uptake of Eu (III) more than 99%. The effect of high-temperature conditions of the repository on the sorption property of bentonite is not significant. The results also suggest that the evolved cement water presents no detrimental effect on the long-term adsorption performance of bentonite even under higher temperature conditions.

Experimental and numerical study on heavy metal contaminant migration and retention behavior of engineered barrier in tailings pond.

Heavy metal pollution is a serious environmental problem globally, particularly in mines and tailings ponds. In this study, based on laboratory and field tests, the migration of heavy metal contaminants in a tailings pond and the retention behavior of a compacted bentonite engineered barrier system on the heavy metal contaminants were analyzed by a numerical simulation. The results demonstrate that the hydraulic conductivity of compacted bentonite is lower than that of the tailings from the laboratory tests. The hydraulic conductivity of the tailings sand decreased with an increase in the dry density and increased with an increase in the concentration of the chemical solution, which could be attributed to the large amounts of fine-grained soil contained in the tailings, according to the grain size distribution test. The hydraulic conductivity of the tailings from the engineering geological survey was between 2.0 × 10-6 and 9.0 × 10-5 m/s, and followed the order: tail coarse sand ＞ tail silty sand ＞ tail medium sand ＞ tail fine silt. The numerical simulation of the seepage could satisfactorily describe the actual working condition of the tailings dam. With the groundwater seepage, the migration range of the heavy metal contaminant in the researched tailings pond reached a maximum of 45 m for 5 years. The retention efficiencies of the 0.2 m engineered barrier against the heavy metal contaminant for 15 and 30 years were 45.4% and 57.2%, respectively. Moreover, the retention efficiency would exceed 87% when the engineered barrier thickness is increased to 0.5 m. The results of model validation show that the calculated results are in good agreement with the measured ones. These findings can provide effective ideas for the prevention and control of environmental pollution in mines and tailings ponds.

Identification and functional characterization of a solute carrier family 15, member 4 gene in Litopenaeus vannamei.

Innate immunity in shrimp is important in resisting bacterial infection. The NF-κB pathway is pivotal in such an immune response. This study cloned and functionally characterized the solute carrier family (SLC) 15 member A 4 (LvSLC15A4) gene in Litopenaeus vannamei. The open reading frame of LvSLC15A4 is 1, 902 bp long and encodes a putative 633-amino acid protein, which is localized in the plasma membrane and intracellular vesicular compartments. Results of the reporter gene assay showed that LvSLC15A4 upregulated NF-κB target genes, including the immediate-early gene 1 of white spot syndrome virus, as well as several antimicrobial peptide genes, such as pen4, CecA, AttA, and Mtk in S2 cells. Moreover, knocked-down expression of LvSLC15A4 reduced pen4 expression in L. vannamei. LvSLC15A4 down-regulation also increased the cumulative mortality of Vibrio parahemolyticus-infected L. vannamei. Furthermore, LvSLC15A4 expression was induced by unfolded protein response (UPR) in L. vannamei hematocytes. These results suggest that LvSLC15A4 participates in L. vannamei innate immunity via the NF-κB pathway and thus may be related to UPR.

Comparison of a novel in situ polymerase chain reaction (ISPCR) method to other methods for white spot syndrome virus (WSSV) detection in Penaeus vannamei.

Penaeus vannamei were experimentally injected with white spot syndrome virus (WSSV) and tested for WSSV at different times post-injection (p.i.) by 1-step polymerase chain reaction (PCR), 2-step PCR, in situ hybridization (ISH) and in situ polymerase chain reaction (ISPCR) in order to compare sensitivity of the methods. With 1-step PCR, 4 of 15 shrimp tested positive for WSSV at 12 h p.i., and all tested positive by 24 h p.i. With 2-step PCR, 13 out of 15 samples tested positive at 2 h p.i. and all were positive by 4 h p.i. Using in situ hybridization, 1 sample tested positive at 18 h p.i. and all were positive by 36 h p.i. With ISPCR, 1 out of 5 samples was positive at 2 h p.i. and all were positive by 8 h p.i. Two-step PCR showed the highest sensitivity, followed by ISPCR, 1-step PCR and ISH. Although ISPCR revealed WSSV in 9 of 10 P. vannamei that tested positive for WSSV using 2-step PCR, none of the shrimp examined showed clinical signs of WSSV infection or detectable WSSV with 1-step PCR. The major infected organs were muscle and the hepatopancreas.

Litopenaeus vannamei NF-κB is required for WSSV replication.

Many viruses can hijack the host cell NF-κB as part of their life cycle, diverting NF-κB immune regulatory functions to favor their replications. There were several reports on the functions of Litopenaeus vannamei NF-κB (LvNF-κB) in White spot syndrome virus (WSSV) replication in vitro. Here, we studied the relationship between LvNF-κB family protein Dorsal (LvDorsal) and Relish (LvRelish) with WSSV replication in vivo. The expressions of LvDorsal and LvRelish were significantly upregulated by WSSV challenge. Virus loads and expression of viral envelope protein VP28 in LvDorsal or LvRelish silencing shrimps were significantly lower than the control shrimps injected with EGFP-dsRNA or PBS after challenge with 1×10(5) copies WSSV/shrimp. In addition to the LvDorsal activation of WSV069 (ie1) and WSV303 promoter that we have reported, LvRelish can also activate WSV069 (ie1) and WSV303 promoter by dual luciferase reporter assays through screening 40 WSSV gene promoters that have putative multiple NF-κB binding sites. The promoter activity of the WSV069 (ie1) by LvDorsal activation was significantly higher than that by LvRelish activation. WSSV replication in LvDorsal, LvRelish or WSV303 silencing shrimps were significantly inhibited. These results indicate that the L. vannamei NF-κB family proteins LvDorsal and LvRelish expressions are significantly activated by WSSV challenge and WSSV replication partially relied on the activations of LvDorsal and LvRelish in vivo.

Identification and function of leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2) in Litopenaeus vannamei.

Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2) is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G) were identified in Litopenaeus vannamei. All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV). Dual-luciferase reporter assays in Drosophila Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of Drosophila and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of L. vannamei upon V. parahaemolyticus but not S. aureus and WSSV infections. The expression of L. vannamei AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.

Analysis of expression, cellular localization, and function of three inhibitors of apoptosis (IAPs) from Litopenaeus vannamei during WSSV infection and in regulation of antimicrobial peptide genes (AMPs).

Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei. LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrioalginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V. alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibriopenaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.

Characterization of four novel caspases from Litopenaeus vannamei (Lvcaspase2-5) and their role in WSSV infection through dsRNA-mediated gene silencing.

Apoptosis plays an important role in white spot syndrome virus (WSSV) pathogenesis, and caspases are central players in apoptosis. Here, we cloned four novel caspases (Lvcaspase2-5) from the Pacific white shrimp Litopenaeus vannamei, and investigated their potential roles in WSSV replication using dsRNA-mediated gene silencing. Lvcaspase2-5 have the typical domain structure of caspase family proteins, with the conserved consensus motifs p20 and p10. Lvcaspase2 and Lvcaspase5 were highly expressed in muscle, while Lvcaspase3 was highly expressed in hemocytes and Lvcaspase4 was mainly expressed in intestine. Lvcaspase2-5 could also be upregulated by WSSV infection, and they showed different patterns in various tissues. When overexpressed in Drosophila S2 cells, Lvcaspase2-5 showed different cellular localizations. Using dsRNA-medicated gene silencing, the expression of Lvcaspase2, Lvcaspase3, and Lvcaspase5 were effectively knocked down. In Lvcaspase2-, Lvcaspase3- or Lvcaspase5-silenced L. vannamei, expression of WSSV VP28 gene was significantly enhanced, suggesting protective roles for Lvcaspase2, Lvcaspase3 and Lvcaspase5 in the host defense against WSSV infection.