Pathogenesis Of Acute Hepatopancreatic Necrosis Disease Research Articles

Gut microbiota of Pacific white shrimp (Litopenaeus vannamei) exhibits distinct responses to pathogenic and non-pathogenic Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), a high-mortality-rate shrimp disease, is caused by specific Vibrio parahaemolyticus (Vp) strains with a plasmid encoding the PirABVp toxins. As a bacterial pathogen, the invasion of AHPND-causing Vp might impose pressure on commensal microbiota in the shrimp gut, while the relationship between the pathogenesis of AHPND and the dysbiosis of gut bacterial communities remains unclear. Here we explored the temporal changes of shrimp gut microbiota in response to AHPND-causing and non-AHPND-causing Vp strains, with the non-infected controls as a baseline of the shrimp gut microbiota. The diversity and composition of bacterial communities from 168 gut samples (covering three treatments at seven time points with eight individuals per set) were investigated using 16S rRNA gene metabarcoding with high-throughput sequencing. The results showed that (i) species diversity of gut bacterial communities declined in Vp-infected shrimp, independent of the strain pathogenicity; (ii) taxonomic compositions of gut bacterial communities were significantly different between shrimp infected by AHPND-causing and non-AHPND-causing Vp strains; (iii) short-term (within 6 hours) compositional shifts in the gut microbiota were found only in AHPND-causing Vp-infected shrimp; (iv) the gut microbiota of AHPND-causing Vp-infected shrimp was enriched with genera Photobacterium and Vibrio, with a decline in Candidatus Bacilliplasma; and (v) functional predictions suggested the loss of normal metabolism due to compositional shifts in the gut microbiota. Our work reveals distinct features of community dynamics in shrimp gut microbiota, associated with pathogenic versus non-pathogenic Vibrio infections, providing a new perspective of the pathogenesis of AHPND. IMPORTANCE Shrimp production is continually threatened by newly emerging diseases, such as AHPND, which is caused by specific Vp strains. Previous studies on the pathogenesis of AHPND have mainly focused on the histopathology and immune responses of the host. However, more attention needs to be paid to the gut microbiota, which acts as the first barrier to pathogen colonization. In this study, we revealed that shrimp gut microbiota responded differently to pathogenic and non-pathogenic Vp strains, with bacterial genera Photobacterium and Vibrio enriched in pathogenic Vp-infected shrimp, and Candidatus Bacilliplasma enriched in non-pathogenic Vp-infected shrimp. Moreover, functional predictions suggested that changes in taxonomic compositions would further affect normal metabolic functions, emphasizing the importance of sustaining an equilibrium in the gut microbiota. Several biomarkers associated with specific microbial taxa and functional pathways were identified in our data sets, which help predict the incidence of disease outcomes.

Microbial signature profiles of Penaeus vannamei larvae in low-survival hatchery tanks affected by vibriosis.

Vibriosis is caused by some pathogenic Vibrio and produces significant mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei larvae in commercial hatcheries. Acute hepatopancreatic necrosis disease (AHPND) is an emerging vibriosis affecting shrimp-producing countries worldwide. Zoea 2 syndrome is another type of vibriosis that affects the early stages of P. vannamei larvae. Although the pathogenesis of AHPND and zoea 2 syndrome is well known, there is scarce information about microbial composition and biomarkers of P.vannamei larvae affected by AHPND, and there is no study of the microbiome of larvae affected by zoea 2 syndrome. In this work, we characterized the microbiome of P. vannamei larvae collected from 12 commercial hatchery tanks by high-throughput sequencing. Seven tanks were affected by AHPND, and five tanks were affected by zoea 2 syndrome. Subsequently, all samples were selected for sequencing of the V3-V4 region of the16S rRNA gene. Similarity analysis using the beta diversity index revealed significant differences in the larval bacterial communities between disease conditions, particularly when Vibrio was analyzed. Linear discriminant analysis with effect size determined specific microbial signatures for AHPND and zoea 2 syndrome. Sneathiella, Cyclobacterium, Haliea, Lewinella, among other genera, were abundant in AHPND-affected larvae. Meanwhile, Vibrio, Spongiimonas, Meridianimaribacter, Tenacibaculum, among other genera, were significantly abundant in larvae affected by zoea 2 syndrome. The bacterial network at the phylum level for larvae collected from tanks affected by AHPND showed greater complexity and connectivity than in samples collected from tanks affected by zoea 2 syndrome. The bacterial connections inter Vibrio genera were higher in larvae from tanks affected by zoea 2 syndrome, also presenting other connections between the genera Vibrio and Catenococcus. The identification of specific biomarkers found in this study could be useful for understanding the microbial dynamics during different types of vibriosis.

Shrimp Lipid Droplet Protein Perilipin Involves in the Pathogenesis of AHPND-Causing Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), caused by a unique strain of Vibrio parahaemolyticus (Vp (AHPND)), has become the world’s most severe debilitating disease in cultured shrimp. Thus far, the pathogenesis of AHPND remains largely unknow. Herein, in Litopenaeus vannamei, we found that a Vp (AHPND) infection significantly increased the expression of lipid droplets (LDs) protein LvPerilipin, as well as promoted the formation of LDs. In addition, the knockdown of LvPerilipin increased the shrimp survival rate in response to the Vp (AHPND) infection, and inhibited the proliferation of Vp (AHPND). Furthermore, we demonstrated that LvPerilipin depletion could increase the production of reactive oxygen species (ROS), which may be responsible for the decreased Vp (AHPND) proliferation. Taken together, our current data for the first time reveal that the shrimp lipid droplets protein Perilipin is involved in the pathogenesis of Vp (AHPND) via promoting LDs accumulation and decreasing ROS production.

Identification and Expression Analysis of an Interacting Protein (LvFABP) that Mediates Vibrio parahaemolyticus AHPND Toxin Action.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus causing AHPND (VPAHPND) is the most serious disease affecting shrimp farming. The PirAvp and PirBvp toxins of VPAHPND are known virulence factors. However, the corresponding target protein in shrimp that mediates their action has not been identified. By screening yeast two-hybrid cDNA libraries from intestine, stomach, and hepatopancreas of Litopenaeus vannamei, the protein with the largest increase in gene expression in shrimp hepatopancreas in response to VPAHPND challenge was identified and designated LvFABP. Analysis revealed high sequence homology of the LvFABP gene and a lipocalin/cytosolic fatty acid binding gene. Yeast two-hybrid pairwise analysis, GST-pull down assay, and far-western blot assay were performed to determine the interaction between LvFABP and PirBvp. LvFABP was able to directly bind to PirBvp. The expression of LvFABP in the hepatopancreas was significantly higher at P23 and P27 developmental stages of L. vannamei. RNA interference (RNAi) of LvFABP reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the intestine, stomach, and hepatopancreas after VPAHPND challenge. We concluded that the LvFABP was involved in AHPND pathogenesis and acted as a VPAHPND toxin interacting protein. This is the first identification of VPAHPND toxin interacting protein from the shrimp digestive system by yeast two-hybrid library screening and were confirmed by in vitro protein interaction verification and in vivo challenge experiments. This study provides novel insight into the contributions of LvFABP towards AHPND pathogenesis in shrimp. The findings could inform AHPND preventative measures in shrimp farming.

Litopenaeus vannamei peritrophin interacts with WSSV and AHPND-causing V. parahaemolyticus to regulate disease pathogenesis

Peritrophins are peritrophic membrane (PM) proteins that can interact with chitin fibers via chitin-binding domains. Peritrophins have essential roles in providing porosity and strength to the PM that lines the shrimp midgut. Acute hepatopancreatic necrosis disease (AHPND), caused by strains of V. parahaemolyticus, is known to initially colonize the shrimp stomach and simultaneously disrupt its structural barriers (e.g., cuticle or epithelial tissues) to reach the hepatopancreas. Although stomach and hepatopancreas were identified as target tissues involved in AHPND pathogenesis, our results indicated that peritrophin in peritrophic membrane has a crucial role in determining not only colonization of AHPND-causing bacteria but also their tissue distribution. As the interaction between LvPeritrophin (LvPT) and WSSV (white spot syndrome virus) is not well understood, we noted that LvPT expression was upregulated in shrimp stomach challenged with either WSSV or AHPND. In an in vitro pathogen binding assay, there was strong binding of recombinant LvPT WSSV and AHPND-causing V. parahaemolyticus, and various bacteria. Furthermore, dsRNA-mediated LvPT silencing inhibited WSSV gene expression and viral genome replication. However, downregulation of LvPT gene expression increased copies of AHPND-causing bacteria in shrimp digestive tract, and facilitated bacterial colonization in stomach. In conclusion, we speculated that LvPT might regulate bacterial colonization during AHPND, whereas in WSSV infection, LvPT silencing favored the host. Although recombinant LvPT had strong binding with WSSV, the precise role of LvPT in WSSV infection needs further investigation. These findings increased our understanding of host-pathogen interactions in AHPND and WSSV infection that can be applied in shrimp aquaculture for developing effective antibacterial and antiviral strategies.

Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor.

Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach.

The PirB toxin protein from Vibrio parahaemolyticus induces apoptosis in hemocytes of Penaeus vannamei

ABSTRACT Acute hepatopancreatic necrosis disease (AHPND) is a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. The Pir toxin proteins secreted by a unique strain of Vibrio parahaemolyticus play an essential role in the pathogenesis of AHPND. At present, most studies on the effects of Pir toxin proteins in shrimp focus on digestive tissues or organs such as hepatopancreas, stomach, etc., with none on the immune organs. In the present study, two recombinant Pir toxin proteins (rPirA and rPirB) of V. parahaemolyticus were expressed with rPirB shown to enter shrimp hemocytes. Employing pull-down and LC-MS/MS analysis, GST-rPirB was found to interact with 13 proteins in hemocytes, including histone H3 and histone H4 and among which histone H4 had the highest protein score. Further analysis using GST pull-down and Far-Western blot analysis revealed that rPirB could interact with histone H4. In addition, using the purified nucleosome protein from Drosophila S2 cells, it was found that PirB protein could specifically bind to histones. When flow cytometry was applied, it was observed that the interaction between PirB and histones in shrimp hemocytes induces apoptosis, which results in the dephosphorylation of Serine 10 in histone H3. Collectively, the current study shows that in addition to its effect on the digestive tract of shrimp, the PirB toxin protein interacts with histones to affect the phosphorylation of histone H3-S10, thereby inducing apoptosis.

In Litopenaeus vannamei, the cuticular chitin-binding proteins LvDD9A and LvDD9B retard AHPND pathogenesis but facilitate WSSV infection

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication.

Development of an indirect Enzyme Linked Immunoassay (iELISA) using monoclonal antibodies against Photorhabdus insect related toxins, PirAVp and PirBVp released from Vibrio spp

An acute hepatopancreatic necrosis disease (AHPND) causes serious losses to the global shrimp industry. The etiologic agent of AHPND is Vibrio spp. carrying a large plasmid which encodes a binary toxin, PirAB. Currently, AHPND is diagnosed by PCR based methods that detect the presences of both pirA and pirB genes. However, the bacterial strains containing the pirA and pirB genes do not always express the binary toxin, resulting in mis-estimation of the virulence of bacterial strains containing pirA and pirB genes. Thus, the immuno based assay (i.e. ELISA) is a promising approach to detect PirAVp and PirBVp. In the present study, a total of forty monoclonal antibodies clones (mAb) against PirAVp (20 mAbs) and PirBVp (20 mAbs) were screened by western blot analysis to select four mAb clones that show the strongest immunoreactivity in indirect ELISA (iELISA). The four selected mAbs (i.e. 1B9 and 5E9 against PirAVp; 7B7 and 7B9 against PirBVp) detected specifically Vibrio spp. causing AHPND. In addition, four selected mAbs were able to detect either PirAVp or PirBVp down to 0.008 ng/μl. A double blind assay using thirty AHPND-infected and six SPF shrimp Penaeus vannamei were analyzed by iELISA to determine the detection sensitivity of the assay. The results showed that iELISA was able to accurately detect 29 out of 30 AHPND infected shrimp. These finding indicated that iELISA is a reliable method to detect PirAVp and PirBVp toxins in infected shrimp and will be a useful tool in AHPND diagnosis and in studying the role of binary toxins in AHPND pathogenesis.

Complete genome sequence analysis of the Vibrio owensii strain SH-14 isolated from shrimp with acute hepatopancreatic necrosis disease.

Vibrios are a group of very important bacterial pathogens in marine aquaculture industry and cause serious aquatic animal diseases, such as shrimp acute hepatopancreatic necrosis disease (AHPND). A new AHPND pathogen, the Vibrio owensii strain SH-14, was isolated from diseased shrimp in Shanghai, China. In this study, to better understand the pathogenesis of AHPND at the genomic level, the genome of the strain SH-14 was completely sequenced and analyzed. The SH-14 consists of two circular chromosomes of 3,689,702bp and 2,430,445bp, and of two plasmids named as pVHvo (69,148bp) and pVHvo-R (78,918bp), respectively. The pVHvo encodes the bi-toxic genes of pirAB, responsible for shrimp AHPND. The whole genomes contain a total of 5703 predicted open reading frames (ORFs), 129 tRNA genes and 37 rRNA genes. The average nucleotide identities (ANIs) between the SH-14 and the other V. owensii strains are all greater than 95%, confirming a new V. owensii strain of the SH-14. The taxonomic affiliation of the SH-14 is also supported by whole-genome alignment and nucleotide identity dotplot analyses. These results pave the way for further study of spread and epidemic of shrimp AHPND.

Acute hepatopancreatic necrosis disease in penaeid shrimp

AbstractAsian countries are the major producers of cultured Penaeid shrimps such as Penaeus monodon and Penaeus (Litopenaeus) vannamei. In recent years, shrimp production has declined due to the emergence of a bacterial disease called acute hepatopancreatic necrosis disease (AHPND). This disease is mainly caused by Vibrio parahaemolyticus, but other Vibrio species are also known to cause AHPND in shrimps. Here, in addition to reviewing recent developments in the field of AHPND diagnosis, host responses to AHPND, the role of microbiota during AHPND infection and the current treatment options for AHPND, we also describe a model of AHPND pathogenesis.

The Rho signalling pathway mediates the pathogenicity of AHPND-causing V.parahaemolyticus in shrimp.

An emerging bacterial disease, acute hepatopancreatic necrosis disease (AHPND), is caused by strains of Vibrio parahaemolyticus with an additional AHPND-associated plasmid pVA1 encoding a virulent toxin (Pirvp ) that damages the shrimp's hepatopancreas. Like other species of Vibrio, these virulent strains initially colonise the shrimp's stomach, but it is not yet understood how the bacteria or toxins are subsequently able to cross the epithelial barrier and reach the hepatopancreas. Here, by using transcriptomics and system biology methods, we investigate AHPND-induced changes in the stomach of AHPND-causing V.parahaemolyticus (5HP)-infected shrimp and identify host molecular mechanisms that might explain how the integrity of the stomach barrier is compromised. We found that the expression of 376 unique genes was differentially regulated by AHPND infection. Gene ontology, protein interaction, and gene-to-gene correlation expression interaction analyses indicated that in addition to the immune system, a number of these genes were involved in cytoskeleton regulation by Rho GTPase. The involvement of Rho pathway regulation during AHPND pathogenesis was further supported by experiments showing that while Rho inhibitor pretreatment delayed the infection, pretreatment with Rho activator enhanced the pathogenicity of 5HP, and both the bacteria and toxin were detected sooner in the hepatopancreas. Further, disruption of the stomach epithelial structure was found in both Rho preactivated shrimp and in 5HP-infected shrimp. Taken together, we interpret our results to mean that Rho signalling helps to mediate AHPND pathogenesis in shrimp.

Acute Hepatopancreatic Necrosis Disease (AHPND) related microRNAs in Litopenaeus vannamei infected with AHPND-causing strain of Vibrio parahemolyticus

BackgroundAcute hepatopancreatic necrosis disease (AHPND) has emerged as a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. Given that several diseases and infections have been associated with microRNAs (miRNAs), we conducted a comparative transcriptomic analysis using the AHPND (VA) and non-AHPND (VN) strains of Vibrio parahemolyticus to identify miRNAs potentially involved in AHPND pathogenesis in Litopenaeus vannamei.ResultsA total of 83 miRNAs (47 upregulated and 36 downregulated) were significantly differentially expressed between the VA and VN challenged groups, while 222 target genes of these miRNAs were predicted. Functional enrichment analysis revealed that the miRNAs target genes were involved in multiple biological processes including metabolic pathways, amoebiasis, Vibrio cholerae infection etc. Finally, interaction network and qPCR (Real-time Quantitative PCR) analysis of 12 potential key AHPND-related miRNAs and their predicted target genes, revealed their possible involvement in modulating several immune-related processes in the pathogenesis of AHPND.ConclusionsWe have shown using comparative transcriptomic analysis, miRNAs and their target genes that are responsive to AHPND V. parahemolyticus infection in shrimp, therefore suggesting their possible role in defense response to AHPND V. parahemolyticus infection.

Transcriptome analysis of the hepatopancreas in Exopalaemon carinicauda infected with an AHPND-causing strain of Vibrio parahaemolyticus

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus carrying toxin-producing plasmid, has led to severe mortalities in multiple shrimp species throughout Asia. In order to understand the immunological response of shrimp to infection by AHPND-causing strain of V. parahaemolyticus (VPAHPND), the transcriptomic profiles of the hepatopancreas from severe AHPND-infected (BS_G), AHPND-survived (KB_G) and non-infected (PBS_G) Exopalaemon carinicauda groups were obtained using HiSeq™ 2500 (Illumina). In total, 525 million high quality clean reads were obtained in nine libraries and de novo assembled into 130,082 unigenes with an average length of 724 bp. Based on sequence similarity, 22.75% unigenes were annotated in the public databases. Comparative analysis revealed that 3733 genes differentially expressed in VPAHPND infection compared with the controls, including 1114 and 3461 unigenes in BS_G vs PBS_G and KB_G vs PBS_G, respectively. A total of 229 differential expressed genes that have high homologies with the known proteins in crustacean species were identified, among which 127 genes are reported potentially related to immune function. We identified relative genes and pathways associated with AHPND pathogenesis and defenses. Our results provide valuable information for further analysis of the mechanisms of shrimp defense against AHPND infection.

AHPND biomarker profile identified by transcriptome sequencing in Litopenaeus vannamei stomach

Acute hepatopancreatic necrosis disease (AHPND) is an emerging (since 2009) disease affecting pacific white shrimp (Litopenaeus vannamei) in Southeast Asia. It is generally accepted that it is caused by Vibro parahaemolyticus (VP), with extra-chromosomal elements that produce a virulent toxin. It was reported that VP may multiply in the shrimp stomach and cause severe cellular damage by releasing virulent toxin into shrimp hepatopancreas. In our pioneering studies, we hypothesized that severe cellular damage may also be due to immune overexpression following VP invasion. In addition to development of therapeutic approaches for AHPND, there is a need to monitor biomarkers of disease progression and host immune responses. In this study, next-generation sequencing-based transcriptome analysis of total RNA from shrimp stomach was performed. A non-virulent VP (S02) strain was used to normalize general responses after bacterial invasion. Shrimp were immersed in a virulent VP (5HP) strain and samples collected at 24 hours post immersion (hpi) and designated AHPND-infected shrimp. Furthermore, samples collected at 1 week post immersion (wpi) were designated AHPND-survived shrimp. For 24 hpi AHPND-infected shrimp, there were a few shrimp with no detectable AHPND toxin and immune overexpression, whereas for 1 wpi AHPND-survived shrimp, all lacked any detectable of AHPND toxin and immune overexpression. Therefore, we inferred that these shrimp may have triggered critical responses that can overcome VP invasion. By comparing these shrimp with severe AHPND-infected shrimp, we identified relative genes and pathways associated with AHPND pathogenesis and defenses. This study documented host gene changes in response to AHPND; furthermore, pathways identified may be key for successfully controlling AHPND.

Characterization and involvement of toll and IMD pathways in AHPND-infected shrimp

Acute hepatopancreatic necrosis disease (AHPND) is emerging as a major disease of shrimp industries worldwide, causing almost 100% mortality at the postlarvae stage. The etiology is Vibrio parahaemolyticus (Vp), a Gram-negative marine bacteria, which must have received a plasmid coding for production of PirA/B, a protein toxin. Based on molecular and histological studies, Vp accumulated in the stomach and secreted PirA/B, which damaged the stomach and hepatopancreas (major AHPND lesions). However, the mechanism by which AHPND kills shrimp remains unclear. Here, we demonstrated that genes of Toll and IMD pathway, Toll, MyD88 and Relish, and its downstream antimicrobial peptides (AMPs) genes, PEN2, 3, 4, anti-lipopolysaccharide factor (ALF), crustin and lysozyme (lyz), were suppressed in stomach. Surprisingly, this phenomenon also occurred in hemocytes (major immune organ of shrimp). Furthermore, most of these genes were overexpressed (particularly in the IMD pathway), in the hepatopancreas, a target organ for AHPND. We subsequently used dsRNAmediated silencing to confirm involvement of Toll and IMD pathway in pathogenesis of AHPND.