Supplementary Material
Materials and Methods
3’- and 5’-RACE PCR with C. salei cDNA
A set of three primers for 3’-RACE and one primer for 5’-RACE PCR was designed based on
conserved regions identified by aligning the sequence of a spider defensin from Argiope sp. [12]
with defensin sequences from other arthropods: Spider def fwd 1, Spider def fwd 2, Spider def
fwd 3, Spider def rev 3 (Table S1). These primers were used for PCR with 1/100th of the
synthesized cDNA of C. salei and the universal primer UPM from the BD SMART™ RACE
cDNA Amplification Kit, taking advantage of the BD SMART sequence incorporated into the
cDNAs. PCR was performed for 5 min at 94°C, followed by 5 cycles of 94°C for 30 s, 63°C for
30 s, 72°C for 2 min, 5 cycles of 94°C for 30 s, 61°C for 30 s, 72°C for 2 min, 5 cycles of 94°C
for 30 s, 59°C for 30 s, 72°C for 2 min, 5 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 2 min,
5 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 5 cycles of 94°C for 30 s, 53°C for 30 s,
72°C for 2 min, 10 cycles of 94°C for 30 s, 51°C for 30 s, 72°C for 2 min, and a final elongation
at 72°C for 7 min. PCR products were run on a 0.7% agarose gel containing 0.5 ng ethidium
bromide /ml. Single bands were cut out using a scalpel, and DNA was extracted from the gel
using a MinElute® Gel Extraction Kit (Qiagen, Switzerland). One third of the extracted DNA was
then used as template in another round of PCR, using the same primers as before. The PCR
program was also the same as before, except that the elongation phases were reduced from 2 min
to 1.5 min. PCR products were then purified using the MinElute® PCR Purification Kit (Qiagen,
Switzerland).
From the sequencing results of these PCR products, further primers for 3’-RACE PCR were
designed: C.salei def fwd 1, C.salei def fwd 2, C.salei def fwd 3 and C.salei sig fwd 1. Also, more
primers were designed for 5’-RACE PCR: C.salei def rev 2, C.salei def rev 3 and C.salei def rev
4 (Table S1). 3’- and 5’-RACE PCRs were performed with these primers and the universal
primer UPM, with the same PCR program as above. PCR products were then purified again using
the MinElute® PCR Purification Kit.
3’- and 5’-RACE PCR with P. pythagoricus cDNA
Each of the following primers was used together with the universal primer UPM for PCR with
1/100th of the synthesized cDNA of P. pythagoricus: Spider def fwd 1, Spider def fwd 2, Spider
def fwd 3, Spider def rev 3, C.salei def fwd 1, C.salei def fwd 2, C.salei def rev 2 and C.salei sig
fwd 1. PCR was performed with the same program described above. Candidate PCR products
were run on a 0.7% agarose gel containing 0.5 ng ethidium bromide /ml. Single bands were cut
out using a scalpel, and DNA was extracted from the gel using a MinElute® Gel Extraction Kit.
Half of the extracted DNA was then used as template in another round of PCR, using the same
primers as before together with the nested universal primer NUP instead of the universal primer
UPM. Products from this “nested” PCR were purified using the MinElute® PCR Purification Kit.
From the sequencing results of these PCR products, two further primers for 3’-RACE PCR were
designed: Polybetes def fwd and Polybetes sig fwd (Table S1). 3’-RACE PCR was performed
with each of these primers together with the nested universal primer NUP using 3/100th of the
synthesized cDNA of P. pythagoricus as template. PCR was performed for 5 min at 94°C,
followed by 5 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 1.5 min, 5 cycles of 94°C for 30 s,
51°C for 30 s, 72°C for 1.5 min, 5 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 1.5 min, 5
cycles of 94°C for 30 s, 49°C for 30 s, 72°C for 1.5 min, 5 cycles of 94°C for 30 s, 48°C for 30 s,
72°C for 1.5 min, and a final elongation at 72°C for 7 min. The PCR products were purified using
the MinElute® PCR Purification Kit.
3’- and 5’-RACE PCR with T. atrica cDNA
The following primers were used in 3’-RACE PCR to screen for defensins in T. atrica hemocytes
together with the nested universal primer NUP: Spider def fwd 1, Spider def fwd 2, Spider def
fwd 3, C.salei def fwd 1, C.salei def fwd 2, C.salei def fwd 3 and Polybetes def fwd. PCR with
1/50th of the synthesized cDNA of T. atrica was performed with the following program: 5 min at
94°C, followed by 5 cycles of 94°C for 30 s, 51°C for 30 s, 72°C for 45 s, 5 cycles of 94°C for 30
s, 50°C for 30 s, 72°C for 45 s, 5 cycles of 94°C for 30 s, 49°C for 30 s, 72°C for 45 s, 5 cycles of
94°C for 30 s, 48°C for 30 s, 72°C for 45 s, 5 cycles of 94°C for 30 s, 47°C for 30 s, 72°C for 45
s, 15 cycles of 94°C for 30 s, 46°C for 30 s, 72°C for 45 s, and a final elongation at 72°C for 7
min . Candidate PCR products were run on a 1.6% agarose gel containing 0.5 ng ethidium
bromide /ml. Single bands were cut out using a scalpel, and DNA was extracted from the gel
using a MinElute® Gel Extraction Kit. Half of the extracted DNA was then used as template in
another round of PCR, using the same primers as before and the following program: 3 min at
94°C, followed by 40 cycles of 94°C for 30 s, 45°C for 30 s, 72°C for 30 s, and a final elongation
at 72°C for 7 min. Products from this “nested” PCR were purified using the MinElute® PCR
Purification Kit.
From the sequencing results of the 3’-RACE PCR products, the primer C.salei def rev 3 was
designated for 5’-RACE PCR together with the universal primer UPM. PCR with 1/33rd of the
synthesized cDNA of T. atrica as template was performed with the following program: 3 min at
94°C, followed by 40 cycles of 94°C for 30 s, 42°C for 30 s, 72°C for 45 s, and a final elongation
at 72°C for 7 min. Candidate PCR products were run on a 1.8% agarose gel containing 0.5 ng
ethidium bromide /ml. Single bands were cut out using a scalpel, and DNA was extracted from
the gel using a MinElute® Gel Extraction Kit and directly used for cloning.
3’- and 5’-RACE PCR with P. reidyi cDNA
The following primers were used in 3’-RACE PCR to screen for defensins in P. reidyi hemocytes
together with the nested universal primer NUP: Spider def fwd 1, Spider def fwd 2, Spider def
fwd 3, C.salei def fwd 1, C.salei def fwd 2, C.salei def fwd 3 and Polybetes def fwd. PCR with
1/100th of the synthesized cDNA of P. reidyi was performed with the following program: 5 min
at 94°C, followed by 5 cycles of 94°C for 30 s, 51°C for 30 s, 72°C for 45 s, 5 cycles of 94°C for
30 s, 50°C for 30 s, 72°C for 45 s, 5 cycles of 94°C for 30 s, 49°C for 30 s, 72°C for 45 s, 5
cycles of 94°C for 30 s, 48°C for 30 s, 72°C for 45 s, 5 cycles of 94°C for 30 s, 47°C for 30 s,
72°C for 45 s, 10 cycles of 94°C for 30 s, 46°C for 30 s, 72°C for 45 s, and a final elongation at
72°C for 7 min. Candidate PCR products were purified using the MinElute® PCR Purification
Kit.
From the sequencing results of the 3’-RACE PCR products, the primer C.salei def rev 4 was
designated for 5’-RACE PCR together with the nested universal primer NUP. PCR with 1/50th of
the synthesized cDNA of P. reidyi as template was performed with the following program: 5 min
at 94°C, followed by 5 cycles of 94°C for 30 s, 51°C for 30 s, 72°C for 30 s, 5 cycles of 94°C for
30 s, 50°C for 30 s, 72°C for 30 s, 5 cycles of 94°C for 30 s, 49°C for 30 s, 72°C for 30 s, 5
cycles of 94°C for 30 s, 48°C for 30 s, 72°C for 30 s, 5 cycles of 94°C for 30 s, 47°C for 30 s,
72°C for 30 s, 15 cycles of 94°C for 30 s, 46°C for 30 s, 72°C for 30 s, and a final elongation at
72°C for 7 min. The PCR products were purified using the MinElute® PCR Purification Kit.
3’-RACE PCR with M. menardi cDNA
The following primers were used in 3’-RACE PCR to screen for defensins in M. menardi
hemocytes together with the universal primer UPM: Spider def fwd 1, Spider def fwd 2, Spider
def fwd 3, C.salei def fwd 1, C.salei def fwd 2, C.salei def fwd 3 and Polybetes def fwd. PCR
with 1/100th of the synthesized cDNA of M. menardi. was performed with the following program:
5 min at 94°C, followed by 5 cycles of 94°C for 30 s, 51°C for 30 s, 72°C for 1 min, 5 cycles of
94°C for 30 s, 50°C for 30 s, 72°C for 1 min, 5 cycles of 94°C for 30 s, 49°C for 30 s, 72°C for 1
min, 5 cycles of 94°C for 30 s, 48°C for 30 s, 72°C for 1 min, 5 cycles of 94°C for 30 s, 47°C for
30 s, 72°C for 1 min, 5 cycles of 94°C for 30 s, 46°C for 30 s, 72°C for 1 min, and a final
elongation at 72°C for 7 min . Candidate PCR products were run on a 0.7% agarose gel
containing 0.5 ng ethidium bromide /ml. Single bands were cut out using a scalpel, and DNA was
extracted from the gel using a MinElute® Gel Extraction Kit. Half of the extracted DNA was then
used as template in another round of PCR, using the same primers and program as before.
Products from this “nested” PCR were purified using the MinElute® PCR Purification Kit.
Table S1. Primers used in this study
3’-RACE primers
Spider def fwd 1
Spider def fwd 2
Spider def fwd 3
C.salei def fwd 1
C.salei def fwd 2
C.salei def fwd 3
C.salei sig fwd 1
Polybetes def fwd
Polybetes sig fwd
5’-GGATTCGGGTGTCCTTTCTGCCA-3’
5’-CCAAGGGGAATGTAACCTTCACTGC-3’
5’-TTCAAACAAACCTGCAAATGCAAC-3’
5’-GGATTTGGATGCCCAKTCAATC-3’
5’-TGTCACAAACACTGCCAAAGTGTT-3’
5’-AGATACAGAGGAGGTTACTGTACCAA-3’
5’-ATGAAGACTGCACATATTCTCCTC-3’
5’-GGTTTTGGATGTCCTTTGAATCAG-3’
5’-ATGAAAGCAGGTCGTGTATTTCTC-3’
5’-RACE primers
Spider def rev 3
C.salei def rev 2
C.salei def rev 3
C.salei def rev 4
5’-GTTGCATTTGCAGGTTTGTTTGAA-3’
5’-AACACTTTGGCAGTGTTTGTGACA-3’
5’-TATCTAACACTTTGGCAGTGTTTGTG-3’
5’-CTTGAGAAAGTTGGTACAGTAACCTCC-3’
Universal primers
UPM
NUP
5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’
5’-AAGCAGTGGTATCAACGCAGAGT-3’
Tissue expression primers
5’-CGTAAGGATTGGTACGATGT-3’
Lycosa S3A fwd
5’-TCCATTCCGTGAAAGTTGGT-3’
Lycosa S3A rev