Yorkie Negatively Regulates the Expression of Antimicrobial Proteins by Inducing Cactus Transcription in Prawns Macrobrachium nipponense

The Hippo signaling pathway controls organ size and immune system in Drosophila and mammals. Yorkie acts as a transcriptional co-activator in the Hippo pathway and cross-talks with other essential pathways. In this study, a Yorkie gene and two Cactus isoforms (designated as MnYorkie, MnCactus-a, and MnCactus-b, respectively) were isolated and characterized from oriental river prawns (Macrobrachium nipponense). Results showed that MnYorkie includes 1620 bp open reading frame and encodes a protein of 539 amino acids (aa). MnCactus-a (377 aa) and MnCactus-b (471 aa) were produced by alternative splicing. MnYorkie and MnCactus were continuously expressed in all selected tissues. Upon Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Vibrio parahaemolyticus stimulation, the mRNA levels of MnYorkie and MnCactus in hemocytes and intestines underwent time-dependent enhancement. RNA interference studies showed that MnYorkie silencing remarkably downregulated the transcription of MnCactus but upregulated the expression of seven immune-related genes. In addition, MnYorkie silencing in vivo decreased the susceptibility of prawns to bacterial challenge. After S. aureus and V. parahaemolyticus infection, the survival rate of prawns increased significantly from 2 to 6 days, which corresponded to the period of MnYorkie knockdown. All these findings suggested that MnYorkie in the Hippo pathway might exhibit remarkable biological roles in the immune defense of M. nipponense by negatively regulating the expression of immune-related genes and promoting the transcription of MnCactus.


INTRODUCTION
Invertebrates, such as insects and crustaceans, rely mainly on various innate defense reactions to combat pathogen infections (1). When foreign pathogens invade, the innate immune system triggers various humoral and cellular activities through signal transduction pathways (2,3). Signal transduction involves the binding of extracellular signaling molecular to cellsurface pattern recognition receptors (PRRs) to change the conformation of the receptor, a phenomenon also known as receptor activation (4). Some of the activated PRRs repress the growth or directly kill pathogens, and most of them transmit corresponding signals into the cell to trigger physiological responses (5). Several signaling pathways in invertebrates have been discovered, such as Janus kinase-signal transducer activator of transcription (JAK/STAT), immune deficiency (IMD), Toll, and Hippo pathways (6,7).
The Hippo pathway was first identified as an evolutionarily conserved signaling mechanism that controls organ size by inhibiting cell proliferation and promoting apoptosis in Drosophila (8,9). In canonical Hippo signaling, upstream stimuli activate the Ste-20 family protein kinase Hippo (Hpo), which then binds to and phosphorylates regulatory scaffold protein Salvador (Sav) (10). The Hpo/Sav complex and Mats (Mob as tumor suppressor) adaptor protein phosphorylate and activate Warts (Wts), a nuclear Dbf2-related family protein kinase (11). The Wts/Mats complex then phosphorylates the downstream target protein Yorkie (Yki), a transcriptional coactivator (12). Phosphorylated Yki is prohibited from entering the nucleus to interact with the transcription factor Scalloped (Sd), thus unable to trans-activate gene targets and is inhibited by Hippo signaling (12,13). The components of Drosophila Hippo pathway are highly conserved in mammals. Hpo, Sav, Wts, Mats, and Yki in Drosophila are orthologs of mammalian Ste-20 kinases Mst1 and Mst2 (Mst), Sav, large tumor suppressor (Lats) 1/2, Mps one binder 1A and 1B (MOB), and Yesassociated protein (YAP), respectively (14,15). Human Mst2, Lastl/2, MOB1, and YAP can rescue the corresponding Drosophila mutants and thus are functionally conserved in mammals (16).
In addition to its role in developing or regenerating tissues, the Hippo pathway regulates the immune system in Drosophila and mammals (17). Its dysregulation has been linked to various human cancers (15,18). In Drosophila, the antibacterial response triggered by Hippo signaling can be enhanced by reducing the expression of Cactus, a Yki target gene (19) and an inhibitor of NF-kB (IkB) in the Toll pathway (20). In the absence of Hippo function, Gram-positive bacteria and fungi increase the lethality of flies. Without inhibition by Hpo, Yki translocates into the nucleus to upregulate Cactus transcription, which normally inhibits the nuclear translocation of NF-kB transcription factors Dorsal and Dorsal-related immunity factor (Dif) and the transcription of antimicrobial peptides (AMPs). Upon activation by Gram-positive bacteria, the Toll-Myd88-Pelle cascade leads to the phosphorylation and degradation of the Cka subunit of the Hippo inhibitory complex, thus eventually releasing Hpo to achieve Yki blockage and antimicrobial effects. Therefore, Hippo signaling enhances NF-kB signaling and AMP expression in Drosophila (21). Most of its components in Drosophila have been cloned in prawns, thereby suggesting the existence of Hippo pathway in these invertebrates. Nevertheless, the role of the Hippo pathway in immune regulation in prawns remains unknown.
As a member of the Palaemonidae family of decapod crustaceans, Macrobrachium nipponense is widely distributed in the freshwater and low-salinity estuarine regions of China, Japan, and South-East Asian countries (22). Prawns live in an aquatic environment full of bacteria, fungi, and other potential pathogens. Understanding the molecular mechanism of their innate immunity will contribute to disease control in prawn aquaculture. In this study, a Yorkie gene and two Cactus isoforms (named MnYorkie, MnCactus-a, and MnCactus-b, respectively) were identified from M. nipponense. Their tissue distributions and temporal response to bacterial stimulation were examined. The biological function of MnYorkie was investigated by detecting the expression of downstream immune-related genes, bacterial clearance activity, and organism survival rate. The findings will provide insights into the immune defense mechanisms of crustaceans.

Experimental Animals
A total of 200 healthy M. nipponense specimens with a weight of 2.5-3.5 g were purchased from an aquatic product market in Nanjing, China and kept in an aerated water tank filled with freshwater. After 10 days of acclimatization, hemolymph was extracted from five prawns and placed in an equal volume of an anticoagulant solution (glucose, 1.47 g; citric acid, 0.48 g; trisodium citrate, 1.32 g; prepared in ddH 2 O and added to 100 mL, pH 7.3). Hemocytes were then isolated through centrifugation at 2000 rpm for 10 min. Heart, hepatopancreas, gills, intestine, and stomach were dissected from the prawns and quickly stored at -80°C until RNA extraction.

Total RNA Extraction and cDNA Synthesis
Total RNA was isolated from the collected samples using the TRIzol Reagent (Invitrogen, USA) in accordance with the manufacturer's protocols. RNA quality was assessed by electrophoresis on 1.5% agarose gel, and RNA concentration was measured by a NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA). In brief, 1 mg of RNA was used to synthesize first-strand cDNA using the PrimeScript ® 1st Strand cDNA Synthesis Kit (Takara, Japan) with an oligo-dT primer. The obtained cDNA was kept at -20°C.

Assay of Bacterial Clearance
In brief, 30 mg of MnYorkie-dsRNA or GFP-dsRNA was injected into the prawns twice. At 24 h post second dsRNA injection, 50 mL of S. aureus or V. parahaemolyticus (3 × 10 7 cells) was injected into the prawns. The hemolymph from 10 prawns was extracted and mixed with anticoagulant buffer at 20 min postbacterial injection. After dilution 1:100/1:1000 with PBS, 50 mL of the hemolymph was loaded on LB agar plates and incubated at 37°C overnight. Colony forming unit (cfu) in the plate was counted. The number of S. aureus or V. parahaemolyticus in prawn hemolymphs was calculated and expressed as cfu mL −1 hemolymph. The assay was repeated in triplicate for all samples.

Prawn Survival Rate
Eighty prawns were divided into four groups (MnYorkie-dsRNA plus S. aureus group, GFP-dsRNA plus S. aureus group, MnYorkie-dsRNA plus V. parahaemolyticus group, and GFP-dsRNA plus V. parahaemolyticus group) to determine whether MnYorkie is involved in its host's antibacterial immune defense. Twenty prawns per each experimental group were injected with 30 mg of MnYorkie-dsRNA or GFP-dsRNA twice and then with 50 mL of S. aureus (3 × 10 7 cells) or V. parahaemolyticus (3 × 10 7 cells) at 24 h after the second injection. The number of dead prawns in these four treatment groups was monitored, and the cumulative survival rate of prawns was calculated and recorded up to 144 h. The experiment was repeated three times.

Sequence Characters and Phylogenetic Analysis of MnYorkie
The full-length MnYorkie mRNA (GenBank Accession No. OL901207) is 2558 bp long consisting of a 75 bp 5′untranslated region (UTR), an 863 bp 3′-UTR, and a 1620 bp ORF encoding a predicted polypeptide of 539 amino acids with a calculated Mw of 58.3 kDa and a pI of 5.29 ( Figure 1A). Conserved domain analysis revealed that MnYorkie contains a PDB domain 3KYS|D of 110 amino acids located at 16-126 in the N-terminal region, two WW (domain with 2 conserved Trp (W) residues) domains located at 156-188 and 248-280, five low complexity regions located at 127-146, 193-236, 294-320, 378-404 and 420-437, and a coiled coil region of 24 amino acids located at 341-364 ( Figure 1B).
The deduced amino acid sequence of MnYorkie was aligned with YAP family members as shown in Figure 1C. MnYorkie showed 75.22% identity to PjYAP1 from Penaeus japonicus; 72.01% to PmYAP1 from Penaeus monodon, and 68.85% to PtYAP1 from Portunus trituberculatus. A phylogenetic tree was constructed based on the amino acid sequences of 22 YAP members using NJ method to evaluate its evolutionary position. MnYorkie was nearly clustered with crustacean YAPs, namely, PtYAP1, PjYAP1, PvYAP1, PmYAP1, HaYAP1-B, and CoYAP1 ( Figure 1D).   Figure 2E). In the phylogenetic tree, MnCactus-a and MnCactus-b were first clustered with MrCactus; then grouped with four shrimp Cactus including PjCactus, PcCactus, PvCactus, and PmCactus; and finally clustered into crustacean Cactus including PcCactus, HaCactus, SpCactus, and CoCactus ( Figure 2F).

Tissue Distributions of MnYorkie and MnCactus
The expression levels of MnYorkie and MnCactus in normal M. nipponense tissues were detected by RT-qPCR ( Figures 3A, B) and SqRT-PCR ( Figure 3C). The single peak on the dissociation curve showed the specific amplifications of MnYorkie, MnCactus, and b-actin. MnYorkie expression was highest in the intestines; relatively high in hepatopancreas, stomach, hemocytes, and gills, and the lowest in the heart. MnCactus mRNA transcript was constitutively expressed in all selected tissues with high expression level in hepatopancreas and gills.

Expression Profiles of MnYorkie and MnCactus Upon Bacterial Challenge
RT-qPCR was used to detect the temporal mRNA expression of MnYorkie and MnCactus in hemocytes and intestines after S. aureus and V. parahaemolyticus challenge. Upon S. aureus challenge, the expression level of MnYorkie in hemocytes was greatly upregulated at 12 hpi and recovered to the control level from 24 hpi to 48 hpi ( Figure 4A); MnYorkie mRNA in intestines was dramatically upregulated at 12 hpi, then downregulated at 24 hpi, and increased from 36 hpi to 48 hpi ( Figure 4B); Upon V. parahaemolyticus challenge, the transcript of MnYorkie in hemocytes decreased at 24 h and then increased to the highest level at 36 hpi ( Figure 4C); Its expression in intestines sharply increased to the first peak at 24 hpi, then decreased at 36 hpi, and reached the maximum level at 48 hpi ( Figure 4D). After S. aureus challenge, the expression of MnCactus in hemocytes increased to its respective highest level at 12 hpi and was then slightly downregulated from 24 hpi to 48 hpi ( Figure 5A); With time, the expression level of MnCactus mRNA in intestines increased gradually and reached the highest level at 48 hpi ( Figure 5B); After V. parahaemolyticus infection, the transcript of MnCactus increased sharply at 12 hpi, then decreased slightly at 24 hpi, increased again at 36 hpi, and returned to normal levels at 48 hpi ( Figure 5C); MnCactus expression in intestines continued to upregulate from 12 hpi to 48 hpi ( Figure 5D). These results revealed the potential role of MnYorkie and MnCactus in prawn immunity.

Gene Knockdown
MnYorkie-dsRNA was synthesized successfully and used to challenge the prawns to further explore the function of MnYorkie in vivo. The relative expression levels of MnYorkie in hemocytes and intestines after MnYorkie-dsRNA interference are shown in in Figures 6A, B, respectively. The most significant effect of MnYorkie was detected at 36 hpi. The silencing efficiency of MnYorkie-dsRNA reached up to 76.88% in hemocytes and 57.72% in intestines. Thus, 36 h after dsRNA injection was selected as the optimum time of MnYorkie knockdown and used for further RNAi experiments.

Bacterial Clearance Activity Upon MnYorkie Knockdown
The bacterial clearance activity of MnYorkie towards S. aureus ( Figure 11A) and V. parahaemolyticus ( Figure 11B) was tested to investigate the in vivo effects of MnYorkie on pathogen invasion. A gradual decrease in bacteria count was detected in all experimental groups. At 20 min post S. aureus or V. parahaemolyticus injection, the number of bacteria retained in the MnYorkie-dsRNA group was lower than that in the control group. This finding indicated a high elimination rate in the MnYorkie-dsRNA group. Meanwhile, no significant difference was detected between the GFP-dsRNA and control groups.

MnYorkie Knockdown Promoted the Survival Rate of M. nipponense
Survival rate analysis was performed to evaluate the significance of MnYorkie to prawns during bacterial infections. The survival rate of prawns stimulated by S. aureus ( Figure 12A) and V. parahaemolyticus ( Figure 12B) presented different variation trends after MnYorkie knockdown. Compared with that of the GFP-dsRNA group, the survival rate of the MnYorkie-dsRNA plus V. parahaemolyticus or S. aureus group increased significantly after 2-6 days. These results suggested that MnYorkie silencing promoted the survival rate of prawns infected with bacteria and reduced their cumulative mortality.

DISCUSSION
The roles of Yorkie/YAP, a transcriptional co-activator protein, in Hippo signaling pathway of insects and mammals have been extensively studied. However, its function in the innate immunity of crustaceans remains to be elucidated. In this study, a Yorkie gene from M. nipponense (MnYorkie) was identified and functionally studied. Sequence structure and homologous phylogenetic analysis revealed that the predicted MnYorkie protein exhibits a classical Yorkie structure. Two 33 amino acid WW domains were found in MnYorkie. Given that one of the outstanding features of this sequence motif is the presence of two conserved tryptophans (W), it was named as the WW domain (26). These domains are small, conserved protein modules that are composed of approximately 40 amino acids (27), found in many different signaling and structural proteins, appears folded into a stable, triple stranded b-sheet, and recognized as proline-containing ligands (28). The same structural features indicated that MnYorkie has a potential transcriptional regulatory function in M. nipponense. As a homolog of IkB, Cactus mediates negative-and positivefeedback regulatory loops of NF-kB via different pathways in invertebrates (29). In Drosophila, Cactus is a target gene of Yorkie in the Hippo pathway (19). Here, two Cactus isoforms (MnCactus-a and MnCactus-b) were cloned and identified from M. nipponense through transcriptomic analysis and Significance was compared between the experimental group and the PBS-injected group at the same time point. Significant differences are indicated with an asterisk at P < 0.05, two asterisks at P < 0.01 or three asterisks at P < 0.001. amplification by PCR. The deduced MnCactus-a and MnCactusb amino acid sequences contained the typical characteristics of IkB members: an N-terminal serine-rich region with conserved lysine and serine residues crucial for signal-dependent degradation (30) and a C-terminal ANK repeat domain (six motifs) that binds to the dimerization domain of NF-kB dimers (31). MnCactus-b also consists of a PEST motif (a region rich in proline, glutamate, serine, and threonine residues) at the Nterminus, which is required for the phosphorylation and intrinsic stability of IkB proteins (32). When a cell receives various stimulus signals, NF-kB is released from IkB and then quickly enters the nucleus to activate the expression of downstream effectors (33). Multiple sequence alignment analysis indicated that MnCactus-a and MnCactus-b showed sequence similarities to other crustaceans Cactus proteins. Full-length analysis using NJ method clustered these two into the shrimp group, suggesting their similar functions. These two MnCactus isoforms were formed by alternative splicing; however, the specific splicing method must be further studied in depth.
The tissue localization of MnYorkie transcripts showed its wide expression in all selected tissues and its abundance in intestines and hepatopancreas. Cactus is widely distributed but has distinguished expression profiles among different animals. In white shrimp Litopenaeus vannamei, LvCactus is highly expressed in the heart and muscles but lowly expressed in hepatopancreas (34). FcCactus is highly expressed in the muscles and hemocytes of Chinese shrimp Fenneropenaeus chinensis (35). In M. nipponense, MnCactus mRNA was highly expressed in hepatopancreas, the center of lipid and carbohydrate metabolism with a crucial role in removing potential microbial pathogens (36). The abundance of MnYorkie and MnCactus in hepatopancreas indicated their potential immune defense functions in M. nipponense.
In Drosophila, Hippo signaling is acutely activated only by Gram-positive bacteria (19). In this study, the Hippo pathway in other crustaceans, such as shrimp, lobsters, crayfish, and crabs, the immune response induced by bacteria could be regulated by various signaling pathways (29). The activity and expression of MnYorkie and MnCactus could be directly or indirectly regulated by feedback loops or other signaling pathways to achieve the homeostasis of immune responses. Signal transduction comprises cascade amplification, dispersion, and regulation. One signaling molecule can activate the transduction of multiple signal pathways. Different combinations of several signals may also cause different responses, and coordinated interactions might occur between signal pathways (37). In Drosophila intestinal stem cells, the Hippo pathway is necessary for stem cell division in response to tissue damage via JAK/STAT and epidermal growth factor receptor signaling pathways (Ren et al., 2010). By inhibiting the activity of the Notch pathway, Yorkie acts as an important repressor in specifying polar cells during oogenesis in Drosophila (38). Therefore, the cross regulation of the Hippo pathway and other essential pathways may also exist in other invertebrate species. In previous research, the MnHippo gene in M. nipponense has been identified and subjected to functional analysis (23). Similar to D. melanogaster Hippo, MnHippo knockdown significantly reduced the expression of multiple immune-associated genes, such as MnALF1, MnALF4, MnCrus5, MnCrus7, and MnLyso2 (23). Another immune protein in the Hippo pathway of M. nipponense, MnMOB1, has also been cloned and characterized. MnMOB1 has a positive effect on the expression of AMPs secreted into the extracellular space to resist microbial invaders (39). AMPs are a group of molecules with Mw usually less than 10 kDa and are the major antibacterial effectors in humoral immunity (29). In crustaceans, AMPs are critical immune responsive genes in the NF-kB pathways, including Toll and IMD pathways. In the present study, the silencing of MnYorkie markedly downregulated the transcription of MnCactus but upregulated the expression of six AMPs, namely, MnALF1, MnALF2, MnALF3, MnCrus4, MnCrus5, MnCrus6, and an immune-related gene MnLyso1. In addition, MnYorkie silencing in vivo decreased the susceptibility of prawns to Gram-positive and -negative bacterial infection. After S. aureus and V. parahaemolyticus challenge, the survival rate of prawns increased significantly after 2 days to 6 days, which corresponded to the period of MnYorkie knockdown. In M. nipponense, MnHippo inhibition promoted the translocation of MnYorkie into the nucleus and then regulated the transcription of MnCactus. The high MnCactus expression prevented the translocation of MnDorsal into the nucleus, thereby reducing the expression of some genes, including AMPs, to induce the antimicrobial state of the host.
In conclusion, a Yorkie gene and two Cactus isoforms (named MnYorkie, MnCactus-a and MnCactus-b) were identified from M. nipponense. MnCactus-a and MnCactus-b were formed by alternative splicing. Upon immune challenge with S. aureus and V. parahaemolyticus, the expression levels of MnYorkie and MnCactus mRNA were upregulated in the hemocytes and

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
YH, data curation, formal analysis, funding acquisition, investigation, methodology, software, supervision, validation, visualization, writing -original draft, and writing -review and editing. QS, formal analysis, software, and visualization. JD, data curation, funding acquisition, and validation. QR, conceptualization, project administration, funding acquisition, resources, and writing -review and editing. All authors contributed to the article and approved the submitted version.

FUNDING
The current study was supported by the National Natural Science Foundation of China (32002423), the Natural Science Foundation of Jiangsu Province (BK20180501), the Fundamental Research this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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