---
cDNA cloning, sequencing and functional domain analysis of part of CqDscam’s
extracellular region
Embryos taken from adult Australia redclaw crayfish were extracted using REzolTM C&T reagent (Protech Technology, Taiwan). cDNAs were synthesized using Superscriptase II (Invitrogen) and Anchor dTv primer (Table S1), according to the instruction manual provided by Invitrogen.
The primers used for cloning part of the CqDscam partial extracellular region were designed based on the conserved extracellular Dscam cDNA sequences of two crustaceans: the white shrimp Litopenaeus vannamei (LvDscam; GenBank: GQ154653.1) and the signal freshwater crayfish Pacifastacus leniusculus (PlDscam; GenBank: HQ596367.1) (Table S1).
The relative positions of the primer sets and their expected amplified regions in the schematic Dscam domain structure are shown in Figure S1A. Using cDNAs from crayfish embryos as templates, PCR reactions were performed using 5 primer sets (F1/R1, F1/R3, F4/R6, F2/R5, F5/R7; Fig. S1 and Table S1). All PCR products were cloned into RBC T&A cloning vector (RBC Bioscience) for sequencing. The sequencing results were aligned by Genedoc software and assembled into the cDNA sequence of the partial CqDscam extracellular region. The
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domain architecture of CqDscam was predicted using the SMART website (Simple Molecular Architecture Research Tool, http://smart.embl-heidelberg.de/).
Tissue tropism of CqDscam mRNA
To investigate the tissue tropism of CqDscam mRNA in adult C. quadricarinatus (body size: approximately 14 cm), 8 tissues from a single crayfish (hemocytes, pleopod, gill, heart, hepatopancreas, intestine, nerve, muscle) were subjected to RNA extraction using REzolTMC&T reagent (Protech Technology, Taiwan). RNAs were then reverse transcribed into cDNAs using Superscriptase II (Invitrogen) and Anchor dTv primer (Table S1). The gene expression of CqDscam in crayfish tissues was analyzed using reverse transcription-polymerase chain reactions (RT-PCR) with the CqDscam-specific primer set Ig1-F/Ig3-R or with the housekeeping gene-specific primer set EF1α-F/ EF1α-R (Table S1).
Immunostaining of Dscam in C. quadricarinatus hemocytes
Hemolymph was collected from adult crayfish into an equal volume of anticoagulant solution (10 mM EDTA in PBS, pH 8.0) using a 23 G needle. The hemocytes in the hemolymph
were then seeded directly onto a coverglass and incubated in 2x L15 medium (Invitrogen) (2x Leibovitz’s 15 medium with 10% FBS, 1% glucose, 0.005% NaCl) for 1 h at 25 °C. After
incubation, the cells were washed three times with PBST (PBS containing 0.2% Tween-20) and fixed by using 4 % formaldehyde. After being permeabilized with cold acetone for 3 min, cells
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were incubated with blocking solution (0.01% goat serum in PBST) for 4 h at room temperature and then incubated overnight at 4 °C with polyclonal rabbit anti-Dscam antibody or a polyclonal mouse anti-VDAC antibody (please see [37] for the anti-VDAC antibody preparation). After washing with PBST, the cells were reacted for 5 h at room temperature either with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Sigma) or else with FITC-conjugated donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories). After another washing with PBST, we used 4’-6’-diamidino-2-phenylindole dihydrochloride (DAPI, Vector Laboratories Inc.,) and Rhodamine phalloidin (Invitrogen) to counterstain the nuclei and cytoskeleton, respectively. The cells were mounted on microscope slides and fluorescence signals were examined using a Carl Zeiss LSM780 confocal laser scanning microscope.
Diversity in the variable Ig domains of the extracellular region of CqDscam
To confirm that CqDscam is a classical Dscam as opposed to a Dscam-like protein, we first checked for the presence of variable regions in the Ig2, Ig3 and Ig7 domains. To do this, we amplified two partial cDNA fragments (Ig1-5; Ig5-9) from crayfish cDNAs using the primer sets F1/R3 and F4/R6, respectively (Fig. S1A, Table S1). The PCR products were cloned using the RBC T&A cloning vector (RBC Bioscience) and over 5 colonies of each of the two PCR products were sequenced. The resulting sequences were aligned using Genedoc software. As
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expected, the hypervariable regions of the CqDscam extracellular region were located at the N-terminal of Ig2, N-terminal Ig3, and the entire Ig7 domain. To further investigate the Ig2 and Ig3 exon diversity in the isoform populations of WSSV super-survivors, partial CqDscam hemocyte cDNA fragments containing the Ig2-3 domains were amplified by RT-PCR using the primer set Ig1-F/Ig3-R (Fig. S1A; Table S1). Individual colonies (n = 55~57) containing the variable Ig2-Ig3 region were randomly selected from the WSSV super-survivors and the control group,. Each clone was sequenced and analyzed using Genedoc software.
Quantitative reverse transcription PCR (qRT-PCR)
Total RNA was extracted from hemocyte samples as described above, and cDNA was synthesized using Superscriptase II (Invitrogen) and Anchor dTv primer (Table S1). For the real-time qRT-PCR, primer sets for CqDscam (Cq-Ds-FN-qF/Cq-Ds-FN-qR), for WSSV VP28 (WSSV-VP28-qF/WSSV-VP28-qR), and EF1α (Cq-EF1α-qF/ Cq-EF1α-qR) were used (Table S1). WSSV VP28 was used as the indicator of WSSV infection status while EF1α was used as the internal control gene. Values of CqDscam expression levels were expressed as 2− ΔΔCt.
Indirect ELISA assays
The protein concentration of each crayfish hemolymph sample was determined using a Bradford protein assay kit (Bio-Rad). Briefly, each microtiter well of a 96-well polystyrene plate was coated with 0.75 μg hemolymph in 100 μl 0.1 M carbonate/bicarbonate buffer, pH 9.6,
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and left overnight at 4 °C. After three washes with PBST to remove unbound antigens, the wells were blocked with blocking buffer (2 % BSA in PBST) for 1 h at room temperature. The wells were washed again with PBST and then incubated with anti-Dscam antibody, anti-WSSV VP28 antibody or pre-immune rabbit serum (control IgG) at room temperature for 2.5 h. The plates were then washed one more time with PBST and a 1:5000 dilution of goat anti-rabbit HRP-conjugated secondary antibody was added. After incubating for 1 h at room temperature,
the enzyme activity signal was developed by the addition of freshly prepared 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate (Sigma). After developing for 10 min in the
dark, the stop solution (1N HCl) was added, and the absorbance of each well was measured at a wavelength of 450 nm. All samples were analyzed in duplicate.
Purification of WSSV virions
WSSV virions were purified based on the methods described by Tsai et al. (38) and Xie et al. (39). Briefly, hemolymph collected from WSSV-infected moribund shrimp was centrifuged
to remove the hemocytes and then kept at -80°C as the virus stock. Crayfish (Procambarus
clarkii) were injected with diluted (1: 500 in PBS) virus stock, and 5 ~ 9 days later, all tissues
except for the organs in the cephalothorax were cut into small pieces and homogenized in TESP buffer (50mM Tris, 5mM EDTA, 1 mM PMSF, 500mM NaCl, pH 8.5)(10 ml/g tissue). After centrifugation for 5 minutes at 3500 × g at 4oC, the supernatant was collected and further
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centrifuged at 30,000 × g for 30 minutes at 4oC. The supernatant and the pink upper layer of the pellet were gently removed, and the remaining gray/white pellet was then re-suspended with TM buffer (50 mM Tris, 5 mM MgCl2, pH 7.5). The purity of the WSSV virion samples was checked using SDS-PAGE and Western blotting, and the integrity of the virions was confirmed by negative staining TEM (Transmission Electron Microscopy).
Statistical analysis
Please see the “Supplemental Information”. A Student t-test was used to statistically analyze differences between groups, and Tukey’s multiple-comparison test (SPSS computer software) was used to evaluate differences among individuals. Statistical analysis of the survival rates in the neutralization assays was performed by the Kaplan Meier estimator.
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Table S1. Primers used in this study
a: Cq - Cherax quadricarinatus; Lv - Litopenaeus vannamei
Primer name Speciesa Primer sequence (5’-3’)
F1 Cq 5’- GACAACCGTGTGGACTTCAGC-3’
F2 Cq 5’- TGAACCAAGGTACACAAT-3’
F4 Cq 5’- CTTGAACATCTCTGCAGTTC-3’
F5 Cq 5’- GATACTACTTGTGTGAAGCTAAC -3’
Ig1-F Cq 5’- CTGATGTTCCCTCCCTTC-3’
R1 Cq 5’- GAGTTGTTGACGACACACAGG-3’
R3 Cq 5’- GAACTGCAGAGATGTTCAAG-3’
R5 Cq 5’- CACCATCATGCGTTCACCATC-3’
R6 Cq 5’- CTTCACGAATTGTGTACCTTG-3’
R7 Cq 5’- GACATAACCAGAGCCTTGAC-3’
Ig3-R Cq 5’- CACGTATTATTAGGGTAC-3’
Ds-NdeI-3481F Lv 5’-CCCATATGAGTTCCACTGAGACTCACCTC-3’
Ds-Xhol-4725R Lv 5’-CCCTCGAGTTCATATTCAGCAACTGAAGA-3’
Ds-NdeI-1027F Lv 5’- CCCATATGGAATTTGGACGCCCAGCA-3’
Ds-stop-HindIII-1773R Lv 5’- CCAAGCTTTCAAGCCTGGCTGTTACGGGC–3’
Cq-Ds-FN-qF Cq 5’- GGACGCCTCCCTATAATGGAA-3’
Cq-Ds-FN-qR Cq 5’- CGGCTCAACTTGTATTCAACAATG-3’
WSSV-VP28-qF 5’-AGTTGGCACCTTTGTGTGTGGTA-3’
WSSV-VP28-qR 5’-TTTCCACCGGCGGTAGCT-3’
Cq-EF1α-qF Cq 5’- GGAGAATTTGAAGCTGGGATTTC-3’
Cq-EF1α-qR Cq 5’- CAACTGCTTCACACCCAAGGT-3’
EF1α-F Cq 5’-ACTGGAGAATTTGAAGCTGGG-3’
EF1α-R Cq 5’-ACTTTGGTTCTGTGCTGTCCATC-3’
Anchor dTv 5’-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3’
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Ig2+Ig3 Protocol III
Figs. 4A&4Ba Figs. 4C - 4E Figs. 5B&5C
1 month 2 months 2 months
PBS WSSV
Figs. 4A&4Ba Figs. 4C - 4E Figs. 5B&5C
1 month 2 months 2 months
PBS WSSV
Figs. 4A&4Ba Figs. 4C - 4E Figs. 5B&5C
1 month 2 months 2 months
PBS WSSV
Table S2. Frequency of occurrence (%) of Ig2-Ig3 combinations in CqDscam from the hemocytes of WSSV super-survivors
a. Experimental animals were the same as those used in the indicated Figure[s].
b: Ig2 and Ig3 variable exon (Vi) numbers are the same as those used in Fig. S2.
c: The number in parentheses indicates the number of clones that encoded the complete Ig2 and Ig3 variable region.
d: Combi. N.: The total number of detected combinations.
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Fig. S1. (A) Schematic diagram showing the locations of the primers used in this study. Primer sequences are listed in Table S1. (B) The amino acid sequence of the partial extracellular region Ig1 – FNIII 4 of CqDscam, which is bracketed by the primers F1 and R7. Black shading indicates the Ig domains while the FNIII domains are boxed. (The FNIII 4 domain was not completely sequenced). The RGD motif (gray shading) is between Ig6 and Ig7.
Fig. S2. Multiple amino acid sequence alignments of the CqDscam variable regions of (A) the N-terminal Ig2 domain, (B) the N-terminal Ig3 domain and (C) the entire Ig7 domain. Using RT-PCR, the cDNA sequences of each isoform were obtained from the hemocytes of PBS- and WSSV-injected crayfish. Vi: assigned variable exon number of each expressed alternative sequence.
Fig. S3. Western blotting was used to confirm the presence of CqDscam in the hemolymph samples analyzed by ELISA in Fig. 4A. After subjecting the hemolymph samples (20 μg/sample) and medium containing the recombinant full-length tail-less LvDscam-20 isoform (rP) (positive control) to SDS-PAGE, the separated proteins were transferred onto a PVDF membrane and probed with (A) anti-Dscam antibody and (B) Control IgG. Sample numbers are the same as those used in Figure 4A. The measured size of the full-length CqDscam from super-survivor #4 (224 kDa) is close to the predicted size of full-length PlDscam (222 kDa). The measured size of
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the recombinant tail-less LvDscam (rP) band (203 kDa) is somewhat bigger than the predicted size of tail-less LvDscam (~175 kDa).
Fig. S4. CqDscam and VP28 protein levels in super-surviving individual crayfish subjected to protocol III for 3 months (see Fig. 1). ELISA assays showed that pooled hemolymph from the three survivors in the WSSV challenge group had significantly lower levels of VP28 compared to the surviving crayfish in the PBS group. Only super-survivor #2 in the WSSV challenge group had a significantly higher level of CqDscam in its hemolymph. (B) A binding assay showed that although- super-survivor #2 had a significantly higher level of CqDscam, the WSSV virus particles did not pull down significantly more hemolymph CqDscam from super-survivors #2 than from the control crayfish. Non-immune rabbit sera (Control IgG) was used to measure the non-specific binding in the ELISA assays. “Pool” represents pooled hemolymph from the PBS or WSSV group. Bars labeled with different letters indicate significantly different values (P< 0.05).
Fig. S5. Western blotting was used to confirm the presence of CqDscam in the hemolymph of the 2-month super-survivors analyzed by ELISA in Fig. 6. Hemolymph samples (20 μg/sample) collected 2 days after the second WSSV challenge and positive control medium containing the recombinant full-length tail-less LvDscam-20 isoform (rP) were separated on SDS-PAGE, transferred onto a PVDF membrane and probed with anti-Dscam antibody. Sample numbers are
59
the same as those used in Figure 6. The indicated sizes of 224 kDa and 203 kDa were measured from the full-length super-survivor CqDscam bands and from the tail-less recombinant LvDscam (rP) band, respectively.
Fig. S6. Western blotting was used to test the sensitivity and specificity of two LvDscam antibodies. After subjecting the recombinant His-tagged full-length tail-less LvDscam isoforms 19 and 20 (see [15]) to SDS-PAGE, the separated proteins were transferred onto a PVDF membrane and probed with the indicated antibodies. Medium: culture medium containing the secreted form of recombinant LvDscam.. Lysate: soluble fraction of Sf9-cells infected with the recombinant baculovirus AcMNPV-LvDscam-19 or AcMNPV-LvDscam-20. Pellet: insoluble fraction of Sf9-cells infected with the same AcMNPV-LvDscams. The FNIII 3 – FNIII 6 antibody performed as well as the commercial anti-His antibody and was therefore selected for use in all of the ELISA and Western blot assays in this study.
Fig. S7. Alignment of the amino acid sequence of the FNIII3-FNIII6 region of PlDscam and LvDscam. Shading is used to indicate the occurrence (black 100%) of identical amino acids.
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F1 R1
F1 R3
F2R5
F4 R6
F5 R7
1 3 4 5 6 7 8 9 1 2 3 4 10 5 6 * AAAA
m7G 2
Cloning
Ig3-R Ig1-F Tissue tropism
Ds-FN-qF Ds-FN-qR Real-time PCR
F4 R6
Isoform analysis
Signal peptide Ig domain (variable regions are shaded) Fibronectin domain Transmembrane domain Stop codon
Key:
Ig3-R Ig1-F
AustraliaredclawcrayfishPacific white shrimp
Dscam antibodies Ds-NdeI-1027F Ds-stop-HindIII-1773R
Ds-NdeI-3481F Ds-XhoI-4725R
A
Figure S1A
61
|---1 DNRVDFSN STGANIHCSV RGHPKPTVVW
---Ig1---|
61 VKADDGTAIG DVPGLRKVLS NGTLMFPPFR AEDYRQEVHA QVYRCQATNP HGTVHSRDVH
|---Ig2--121 VRAVVHQDYM TDVSLEYVIL GNCAILKCNI PSFVADFVSV QAWLTDKGQT YYPSGNYDGK
---|
181 YLVLPSGELH IRSVSSEDGF KSYKCRTVHR LTQETRLSAT AGRLVISEAL AASAPKFPTR
|---Ig3---241 DRVSTDRQED TSFSLLCPAQ ADPPPHTRWF KFSENGRKAP VELGDRVKQV GGTLIIREAK ---| |---301 VEDSGKYLCV VNNSVGGESV ETVLTVTAPL SAQVEPSVQT VEFGRPATFT RTYRGNPVKS
---Ig4---|
361 VSWFKDGTPI NHKEAVLRID TVGREDKGMY QCFVRNDQES AQATAELKLG GRFEPPQLTY
|---Ig5---421 TFETSTLQPG PSVYLKCVAA GNPTPEITWE LDGTRLSNSE RMQVGQYVTV NGEAVSHLNI ---| |---481 SAVHTNDGGL YACVASSTVG SVKHAARLNV YGLPYIRPMD KVAVVAGENM VVHCPVAGYP
---Ig6---|
541 IDSIVWEKNG RMLPINRRQK TFPNGTLIVE AVERNSDQGR YTCVARNSQG YTARGDLDVQ
|---Ig7---601 VMEKPEILPF EFPSEVKEGQ LLQVSCTVTT GDDPVTIQWY KEDIPLASSS KFLINKVDSK ---| |---661 MDFLILRDVG SDHTGTYTCL AFNPFGRQRF SAQLWVKVPP RWIVEPTDKA FALGSDARLE
---Ig8---721 CKADGFPRPS LGWKKAAGRT PGDYRDLGVS NPNVKVTDDG TLQIGNIQKS HEGYYLCEAN ---| |---781 NGIGAGLSTV IYVRVQAPPQ FKIQYRNQTA SRGDDAVLEC EAEGETPIGI LWSKNKHSIE ---Ig9---| |-841 PSNEPRYTIR EEMRGGGVHS SLSIKTTDRS DSAVYTCVAT NAFGSADTNI NLIIQEHPEQ
---FNIII1---901 PNSLKVLDKS GRSVELSWTP PYNGNSPITR YIVEYKLSRR NWDSDGERMM VPGDQNMAAV ---| |---961 LDLRPATTYH LRIVARNEIG DSGPSDTVTI ITAEEAPSGP PRDLKVEAVD QSSLRVTWKP
---FNIII2---1021 PLREEWNGDI QGYQVGYRLA SSNNSYVYET VEFSKEMGKE HHLVISKLNI YTEYAVVVSA ---| |---1081 FNKIGQGPKT DEIRAYTAEG TPQQPPQDVT CTTLTSQTIR VSWSSPPLET VQGVIKGYKV
----FNIII3---|
1141 IYGPSDTWYD EESKDTKITG STETRLHGLQ KYTNYSLQVL AFTSGGEGVR SQPIHCQTDQ
|---FNIII4---1201 DIPESPTSVK ALVM
Figure S1B
B
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Alignment of N-terminal Ig2 alternative regions
Vi
Figure S2A
A
63
Alignment of N-terminal Ig3 alternative regions
Vi
64
Alignment of N-terminal Ig7 alternative regions
Vi
C
Figure S2C
65