Cloning and Characterization of Two Toll Receptors (PcToll5 and PcToll6) in Response to White Spot Syndrome Virus in the Red Swamp Crayfish Procambarus clarkii

Toll/Toll-like receptors are key components in the innate immune responses of invertebrates. In this study, we identified two novel Toll receptors (PcToll5 and PcToll6) from the red swamp crayfish Procambarus clarkii. The complete cDNA sequence of PcToll5 is 4247 bp, encoding a 1293 amino acid polypeptide. The full-length 4688 bp PcToll6 encodes a putative protein of 1195 amino acids. Quantitative RT-PCR analysis indicated that PcToll5 and PcToll6 were constitutively expressed in all tissues studied. The highest expression levels of PcToll5 and PcToll6 were found in the intestine and gills, respectively, and were significantly upregulated from 24 to 48 h during white spot syndrome virus (WSSV) challenge. siRNA-mediated RNA interference results showed that PcToll5 and PcToll6 might regulate the expression of anti-lipopolysaccharide factors (PcALF2 and PcALF3) in vivo. Overexpression of PcToll5 and PcToll6 in Drosophila Schneider 2 (S2) cells activated the transcription of Drosophila antimicrobial peptides, including drosomycin (Drs), metchnikowin (Mtk), and attacin A (AttA), and shrimp Penaeidin-4 (Pen4). These findings provide significant information that PcToll5 and PcToll6 may contribute to host immune defense against WSSV in P. clarkii.


INTRODUCTION
The red swamp crayfish Procambarus clarkii is one of the most important farmed freshwater crayfish species worldwide. However, the sustainable culture of crayfish in China has been hindered by disease outbreaks caused by white spot syndrome virus (WSSV) (Maeda et al., 2000). White spot syndrome (WSS) caused by WSSV is the common destructive virus-induced disease (Arts et al., 2007). Similar to other invertebrates, giant freshwater prawns lack an acquired immune system and relies mainly on their innate immunity to fight against invading foreign microbes (Medzhitov and Janeway, 2000a,b). Therefore, the innate immunity of this species in response to pathogen invasion should be studied (Buchmann, 2014). The innate immune system constitutes the first barrier of defense against pathogen invasion in crustaceans and has been conserved throughout the evolutionary process. Upon infection, host germ line-encoded pattern-recognition receptors (PRRs) identify and bind to pathogen-associated molecular patterns (PAMPs) located on the surfaces of microorganisms to trigger multiple downstream signaling pathways Medzhitov and Janeway, 2002;Huang et al., 2013). Scholars have studied several PRRs, such as Toll or Tolllike receptors (TLRs), RIG-like receptors, NOD-like receptors, and C-type lectins (Christophides et al., 2004;Huang et al., 2018). Diverse PRRs react with specific PAMPs, exhibit different expression patterns, activate specific signal pathways, and lead to different anti-pathogen responses .
As one of the most widely studied PRRs, TLRs are involved in pathogen recognition and activation of immune responses Medzhitov, 2007). The first Toll from Drosophila melanogaster is a necessary gene product for embryonic dorsoventral polarity development (Leulier and Lemaitre, 2008;Valanne et al., 2011). This Toll also sense microbial pathogens, such as mammalian TLRs (Lemaitre et al., 1996;Akira et al., 2006). To date, studies have identified 1 Toll-like protein in Caenorhabditis elegans (Tenor and Aballay, 2008), 9 Toll proteins in Drosophila (Valanne et al., 2011), and 10-12 TLRs in mammals (Roach et al., 2005;Akira et al., 2006). A large number of Toll proteins from crustaceans have been widely investigated. In shrimp, Tolls have been studied in Fenneropenaeus chinensis, Penaeus monodon, Marsupenaeus japonicus, Litopenaeus vannamei, and P. clarkii (Arts et al., 2007;Yang et al., 2008;Mekata et al., 2008;Wang et al., 2012Wang et al., , 2015. In the giant tiger shrimp P. monodon, a Toll homolog was expressed in the intestine, gills, and hepatopancreas and could be involved in defense against pathogens (Arts et al., 2007). A Toll receptor from the Chinese shrimp F. chinensis was also expressed in different tissues; the expression was the highest in the lymphoid organ and regulated after Vibrio anguillarum or WSSV stimulation (Yang et al., 2008). A new type of Toll receptor gene was found to be expressed by 76-fold higher than that the control stimulated with peptidoglycan at 12 h in the lymphoid organ of the kuruma shrimp Marsupenaeus japonicus (Mekata et al., 2008). Three different Tolls (Toll1-3) have been studied in the white leg shrimp L. vannamei based on their protein similarities; all these Tolls met the challenges with Vibrio alginolyticus or WSSV (Wang et al., 2012). In the freshwater crayfish P. clarkii, PcToll was upregulated in different tissues after challenge with V. anguillarum or Staphylococcus aureus and was found to be involved in regulation of the expression of antimicrobial peptides (AMPs), including crustins (Cru1 and Cru2), anti-lipopolysaccharide factor 2 (ALF2), and lysozyme 1 (Lys1) . Silencing P. clarkii Toll (PcToll3) influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of the Drosophila Toll signaling pathway (Lan et al., 2016). In crabs, Toll reporters were identified in three crab species, one in Scylla paramamosain (Lin et al., 2012), two in Eriocheir sinensis (Yu et al., 2013), and three in Portunus trituberculatus (Zhou et al., 2015); these reporters were responsive to bacterial pathogens or PAMPs.
In this study, the complete cDNA sequences of two novel Tolls (PcToll5 and PcToll6) from P. clarkii were identified and their responses to WSSV challenge were investigated. This research will be potentially helpful in understanding the innate immune defense of economically important crayfish.

WSSV Challenge and Tissue Collection
One hundred healthy red swamp crayfish (about 15 g each) were obtained from an agricultural market in Nanjing (Jiangsu Province, China). They were acclimated in fresh water in laboratory tanks at 25 • C for a week before the experiments. Hemolymph was collected from at least five crayfish, mixed with 1/3 volume of anticoagulant buffer (10% sodium citrate, pH 7.0) containing 200 mM phenylthiourea, and centrifuged at 800 g at 4 • C for 10 min to isolate hemocytes. Five tissues including heart, hepatopancreas, gill, stomach, and intestine were quickly collected. For the viral challenge experiments, crayfish were divided into two groups (20 crayfish in each group). Each crayfish in group 1 was challenged with 100 µl of WSSV (10 5 copies/mL). Each crayfish in group 2 was injected with 100 µl of PBS and used as blank control . At 0, 24, 36, and 48 h post injection (hpi), the intestines were randomly extracted from five crayfish of each group.

Total RNA Extraction and First-Strand cDNA Synthesis
Total RNA samples from different tissues and the intestine of WSSV-challenged crayfish were extracted in accordance with the manufacturer's instructions (Spin-column, BioTeke, Beijing, China). The 5 and 3 cDNA sequences for the rapid amplification of cDNA ends (RACE) were synthesized using the intestine total RNA as template to obtain the full lengths of PcToll5 and PcToll6 genes. First-strand cDNA (5 cDNA and 3 cDNA) was synthesized using 5 -CDS primer A (5 -T 25 VN-3 ), SMARTer IIA oligo (5 -AAGCAGTGGTAT CAACGCAGAGTACXXXXX-3 ), 3 -CDS primer [5 -AAGCA GTGGTATCAACGCAGAGTAC(T)30VN-3 ], and a Clontech SMARTer TM RACE cDNA Amplification kit from Takara (Dalian, China). First-strand cDNA synthesis of different samples for qRT-PCR analysis was performed using the PrimeScript R First-strand cDNA Synthesis Kit (Takara, Dalian, China) with the Oligo dT Primer .
cDNA Cloning of PcToll5 and PcToll6 Two expressed sequence tags (ESTs) in P. clarkii similar to TLR genes were obtained from our previous high-throughput transcriptome data. On the basis of the original EST sequences, the following gene-specific primers were designed to clone the full-length cDNAs of PcToll5 and PcToll6, respectively: (PcToll5-F: 5 -CGCCTGTGAGGTGTGACCACTATGT-3 , PcToll5-R: 5 -ACATCCAGAACCACCAGGCGAATAAGC-3 ; and PcToll6-F: 5 -GTGTCGTTTTGAGTTCCGTTCCGCCC-3 , PcToll6-R: 5 -ACCAATCGGTGTTGTAGGTCCGCAGC-3 ). The Advantage 2 PCR Kit from Takara (Dalian, China) was used for gene cloning. The PCR amplification conditions were as follows: five cycles at 94 • C for 30 s and 72 • C for 3 min; five cycles at 94 • C for 30 s, 70 • C for 30 s, and 72 • C for 3 min; and 20 cycles at 94 • C for 30 s, 68 • C for 30 s, and 72 • C for 3 min . The PCR products were cloned into the pEASY R -T1 vector (TransGen Biotech) and sequenced (Invitrogen).

Bioinformatics Analysis
Online BLAST program 1 was used to search the homology comparisons of nucleotide and amino acid sequences. cDNA translation, pI analysis, and molecular mass prediction were performed by ExPASy. 2 Domain and signal peptide predictions were conducted by SMART 3 . Multiple protein sequence alignment was performed using MEGA 5.05 and analyzed on GENEDOC software. MEGA 5.05 was utilized to produce phylogenetic trees, and NJ (Neighbor-Joining) method was applied to phylogenetic analysis (Kumar et al., 2008).

Dual Luciferase Activity Assay in S2 Cells
The expression vector for the full-length PcToll5 or PcToll6 was constructed using pAc5.1/V5-His B (Invitrogen, United States). The PCR products were amplified with the specific primers (PcToll5-pAc-F: 5 -CCCGGATCGGGGTACCATGCTCAGCCG CTTGGAGGCCCTTG-3 and PcToll5-pAc-R: 5 -TTCGAACCG CGGGCCCTCTAAAGACGGCATTGCTCTGCGT-3 ; PcToll6-pAc-F: 5 -CCCGGATCGGGGTACCATGCTCCAGGATGTGAC AGTTCTG-3 , and PcToll6-pAc-R: 5 -TTCGAACCGCGGGC CCTCAGTGTCGCCCCATTTAAGATAGG-3 ). After digestion with Kpn I and Apa I (Takara, Dalian, China), the PCR products were cloned into pAc5.1/V5-His B. The positive clones were sequenced to ensure correct insertion. On account of the unavailability of a crayfish cell line, Drosophila Schneider 2 (S2) cells (Invitrogen, United States) were used to explore the activation of AMP transcription of PcToll5 and PcToll6. S2 cells were cultured in Drosophila serum-free medium SDM (Invitrogen, United States) augmented with 10% fetal bovine serum (Invitrogen) at 27 • C. The cells were seeded overnight for DNA transfection, and the plasmids were transfected using the Cellfectin II reagent (Invitrogen). Dual-luciferase reporter assays were carried out by transfecting S2 cells in 96-well plates (TPP, Switzerland) by using 0.3 g of expression plasmids, 0.2 g of reporter gene plasmids, and 0.02 g of pRL-TK renilla luciferase plasmid (Promega, United States) as control. The reporter gene plasmids were constructed using the promoter sequences of the following genes: L. vannamei AMP penaeidin-4 (Pen4), Drosophila AMPs, metchnikowin (Mtk), drosomycin (Drs), and attacin A (AttA). All the assays were performed in triplicate. At 48 h post-transfection, firefly and renilla luciferase activities were measured on the Dual-Luciferase Reporter Assay System (Promega, United States) .

Tissue Distribution and Expression Profiles of PcToll5 and PcToll6
PcToll5 and PcToll6 were expressed in all tissues studied. The highest expression levels of PcToll5 and PcToll6 were found in the intestine and gills, respectively (Figure 3). The expression profiles of PcToll5 and PcToll6 in the intestine or gills were further examined after WSSV challenge. PcToll5 and PcToll6 cDNAs were significantly upregulated in different tissues after challenge with WSSV but showed no significant change in their expression in the PBS-treated group (Figure 4). The highest expression of PcToll5 after challenge with WSSV in the intestine and gills was found at 36 and 48 h post injection, respectively. The PcToll6 expression reached the highest level at 36 h in the intestine and gills. Hence, PcToll5 and PcToll6 might be involved in anti-WSSV immune responses.    PcToll5 and PcToll6 Affect the Transcription of ALFs The expression of PcToll5 or PcToll6 in the corresponding siRNA interference group was significantly knocked down in the intestine of P. clarkii at 36 h compared with that in the WSSV group. Control siRNA-scrambled did not affect the gene expression, indicating that Toll-siRNA is highly specific to PcToll5 or PcToll6. When PcToll5 or PcToll6 expression was FIGURE 4 | Analysis of PcToll5 (A,B) and PcToll6 (C,D) expression in intestine and gills from crayfish challenged with WSSV using qRT-PCR methods. GAPDH was used as an internal control. Asterisks indicate significant differences ( * * * P < 0.001, * * P < 0.01, * P < 0.5) compared with values of the control.

Overexpression of PcToll5 and PcToll6 in Drosophila S2 Cells
FIGURE 5 | Activation of shrimp (Pen4) and Drosophila (Mtk, Drs, AttA) AMPs by over-expression of PcToll5 (A) or PcToll6 (B) in Drosophila S2 cells. Significant statistical differences ( * * P < 0.01, * P < 0.5) are indicated by asterisks. All data are representative of three independent experiments. The bars indicate the mean 6 SD of the luciferase activity (n = 3).
Frontiers in Physiology | www.frontiersin.org inhibited, the ALF expression was tested through qRT-PCR analysis. As shown in Figure 6, two ALFs (PcALF2 and PcALF3) were significantly upregulated 36 h after the WSSV challenge. However, knocked down of PcToll5 or PcToll6 inhibited the transcription of these two ALFs.

Expression of PcALF2 and PcALF3
During WSSV Infection PcALF2 and PcALF3 genes were characterized, and their expression patterns were determined in the intestine of crayfish to evaluate their roles during WSSV infection. As shown in Figure 7, the mRNAs of PcALF2 and PcALF3 were upregulated after WSSV infection at several time points (24, 36, and 48 h). By contrast, the expression of ALFs did not obviously change after PBS challenge.

DISCUSSION
Toll receptors are key components in the innate immune responses of invertebrates. Different types of Tolls have been found in several crustacean species (Arts et al., 2007;Mekata et al., 2008;Yang et al., 2008;Lin et al., 2012;Wang et al., 2012Wang et al., , 2015Yu et al., 2013;Zhou et al., 2015;Lan et al., 2016;Huang et al., 2017). In our previous work, four Toll receptors (PcToll, PcToll2, PcToll3, and PcToll4) were identified in the red swamp crayfish P. clarkii on the basis of sequence similarities and phylogenetic relationships Lan et al., 2016;Huang et al., 2017). In the current study, for the first time, two novel Tolls (PcToll5 and PcToll6) were characterized in P. clarkii. Based on the phylogenetic analyses, PcToll5 and PcToll6 exhibited high similarity to crab Toll protein found in P. trituberculatus and shrimp Toll proteins in M. rosenbergii, P. clarkii, and L. vannamei and clustered on one branch. PcToll5 and PcToll6 contain more than 20 LRR-related motifs, a transmembrane region, and a TIR domain. LRRs are found in more than 2000 proteins from viruses to eukaryotes (Enkhbayar et al., 2004). Most LRRs are composed of 2-45 motifs, which contain 20-30 amino acids in length and provide a structural framework of protein-protein interactions (Kobe and Kajava, 2001;Enkhbayar et al., 2004).
Proteins containing LRRs play a significant role in a number of biological processes, such as signal transduction, DNA repair, cell adhesion, recombination, RNA processing, transcription, disease resistance, apoptosis, and immune response (Rothberg et al., 1990). LRRs are accompanied by cysteine-rich domains: an N-terminal LRR domain and a C-terminal LRR domain. Members of the Toll family are type I transmembrane receptors, which are characterized by an intracellular 200 residue domain with interleukin-1 receptor (IL-1R) and a Toll/IL-1R homologous region (TIR). The TIR domain is essential for Toll proteins and necessary for downstream signal transduction (Kawai and Akira, 2010). This domain is highly conserved not only among different TLRs of one species but also among different animal species (Werling et al., 2009).
PcToll5 and PcToll6 exist in different tissues of crayfish. A high expression of PcToll5 or PcToll6 was observed in the intestine or gills. The intestine provides an active environment for a variety of microbes, including pathogens, because of its digestion and absorption functions (Huang and Ren, 2015). The gills are frequently exposed to the environment because they are relevant for water and air exchange (Huang X. et al., 2016). The different Tolls in L. vannamei were constitutively expressed in all tested tissues (Wang et al., 2012). Tolls identified in three crab species were also widely expressed in few tissues (Lin et al., 2012;Yu et al., 2013;Zhou et al., 2015). Our investigation on the anti-WSSV functions of PcToll5 and PcToll6 showed that WSSV upregulated the expression of PcToll5 and PcToll6 in the intestine or gills after 24, 36, and 48 h of viral challenge. The FcToll expression in the lymphoid organ of F. chinensis was downregulated at early periods after WSSV challenge (Yang et al., 2008). Hemolytic PcToll3 transcription was upregulated 12 h after V. parahemolyticus injection or 24 h post WSSV challenge (Lan et al., 2016). WSSV also upregulated the expression of MrToll in the gills of M. rosenbergii after 24, 36, and 48 h of viral challenge (Feng et al., 2016). Another MrToll was enhanced 3-12 h after Aeromonas caviae stimulation and decreased to basal levels at 24 h post challenge (Srisuk et al., 2014).
Increasing lines of evidence have indicated that the crustacean Toll signaling pathway exerts anti-bacterial, anti-fungal, and FIGURE 7 | Analysis of PcALF2 (A) and PcALF3 (B) expression in intestine from the crayfish challenged with WSSV using qRT-PCR methods. Asterisks indicate significant differences ( * * * P < 0.001, * * P < 0.01) compared with values of the control.
anti-viral functions by regulating the expression of immunerelated genes Lan et al., 2016;Feng et al., 2016). AMPs are one of the major constituents of the invertebrate innate immune system and function as the front line of host defense against microbial infection (Silva et al., 2013). Crustacean AMPs include several families such as Crus, ALFs, and Lys (Lin et al., 2015). Most research focused on the anti-bacterial function of ALFs, which were reported to be involved in anti-viral defense. Pacifastacus leniusculus ALF interfered with WSSV replication and effectively protected crayfish from WSSV infection (Liu et al., 2006). P. monodon ALFPm3 exhibited anti-WSSV activity by interacting with the envelope protein WSSV189 and other WSSV structural proteins (Tharntada et al., 2009;Suraprasit et al., 2014). nLvALF1 with its SNP polymorphisms participated in defense against WSSV in L. vannamei (Liu et al., 2014). In the present study, when PcToll5 or PcToll6 was silenced, the expression levels of PcALF2 and PcALF3 were significantly suppressed after WSSV challenge; hence, PcALF2 and PcALF3 might be regulated by the Toll-mediated signaling pathway. In P. clarkii, PcToll was involved in regulation of Cru1, Cru2, ALF2, and Lys1 expression . PcToll3 silencing influenced the expression of Cru1 and Lys1 after Vibrio challenge (Lan et al., 2016). In the giant freshwater prawn M. rosenbergii, ALF2, ALF3, ALF4, and ALF5 were regulated by MrToll in the gills during WSSV challenge (Feng et al., 2016). In the present work, overexpression of PcToll5 or PcToll6 in Drosophila S2 cells was conducted to determine the roles of PcToll5 and PcToll6 in AMP expression. The results also implied that PcToll5 and PcToll6 activated the transcription of AMPs such as AttA, Mtk, Drs, and Pen4. Based on RNAi and overexpression assay, PcToll5 and PcToll6 played important roles in anti-WSSV immune defense by regulating ALF gene expression.

CONCLUSION
Two novel Tolls (PcToll5 and PcToll6) from P. clarkii were cloned and characterized. PcToll5 and PcToll6 were broadly expressed in all tested tissues of crayfish, and their transcription was induced by WSSV challenge. Overexpression and RNAi experiments showed that PcToll5 and PcToll6 could regulate the expression of AMPs. All these results suggest that PcToll5 and PcToll6 may participate in innate immunity against pathogen infection. However, further studies are required to clarify the specific function and immune mechanism of PcTolls in crayfish immune defense.

ETHICS STATEMENT
We declare that appropriate ethical approval and licenses were obtained during our research.

AUTHOR CONTRIBUTIONS
YH and YC carried out the experiments, contributed reagents and materials. YH, KH, and QR designed the experiments and analyzed the data. YH and QR wrote the manuscript. All authors gave final approval for publication.