1
Electronic Supplementary Material (ESM)
2
accompanying
3
“Invasive ants carry novel viruses in their new range and form reservoirs for an existing
4
pathogen” by Sébastien et al. (Biology Letters)
5
6
ESM Methods
7
8
Viral extraction method using Invitrogen iPrep kit
9
RNA was extracted from two Argentine ant samples from Wellington (New Zealand). Each
10
sample included 30 ants homogenised using pestles in Eppendorf tubes containing 400 µL
11
iPrep PureLink Virus kit lysis buffer (Life Technologies, Carlsbad, CA, USA). 400 µL of
12
molecular grade water and 50 µL proteinase K was then added to each sample and incubated
13
at 50°C for 1 hour, followed by 96°C for 5 minutes. Samples were then centrifuged at 12,000
14
× g for 5 minutes. The supernatant was removed and used in the extraction along with the
15
iPrep Purelink Virus kit (Life Technologies, Carlsbad, CA, USA), eluting into 50 µL of RT-
16
PCR molecular grade water (Ambion, Austin, TX, USA).
17
18
Viral extraction method using Thermo Scientific GeneJET kit
19
RNA was extracted from Argentine ant samples using a GeneJET Viral DNA & RNA
20
Purification Kit (Thermo Scientific, Waltham, MA, USA) following modified protocol. Ants
21
from each sample were homogenised using pestles in Eppendorf tubes containing 250 µL
22
GeneJET Viral DNA & RNA Purification Kit lysis buffer, and then 5 µL RNA carrier and 50
23
µL protein K were added. Samples were incubated for 1 hour at 56°C. Samples were briefly
24
centrifuged, before the supernatant was removed and used in the standard manufacturer’s
25
protocol. Amplicons were visualized by gel electrophoresis and purified using USB ExoSAP-
1
26
IT (Affymetrix). The purified amplicons were sent for Sanger sequencing on a capillary
27
sequencer at Massey University Genome Service (New Zealand).
28
29
Active replication of DWV and LHUV-1 using modified RT-PCRs
30
To specifically amplify the negative strand of DWV and LHUV-1 (contig n6409), tag primers
31
were used in a modified one step RT-PCR as described by Yue et al [1]. A pool containing an
32
equal amount of RNA from each positive site from one country was created for each country.
33
The 10-nucleotide long tags for the primers were generated by BARCRAWL [2] using the
34
default settings of the program and one pair of tags was selected for each pair of primers
35
(ESM Table 1). A modified one step RT-PCR was realized using a SuperScript III One Step
36
RT-PCR system with Platnium Taq DNA polymerase kit (Life Technologies). Reaction mixes
37
consisted of a 24.5 µL mix containing ~20 ng RNA, 1X Reaction mix (final concentration),
38
0.8 µM of reverse tagged primer (either tag- DWVrtR or tag-n6409-A1055R – final
39
concentration), 5.5 µL ddH2O and 1 µL SuperScript® III RT/Platinum® Taq Mix. After the
40
reverse transcription step at 45°C for 30 minutes, 0.8 µM forward tagged primer (tag-
41
DWVrtF or tag-n6409-A1055R) was added to obtain a final 25.0 µL mix. The PCR thermal
42
cycling continued with an initial denaturation step at 94˚C for 2 minutes, followed by 35
43
cycles of 94˚C for 15 seconds, 55˚C for 15 seconds and 68˚C for 30 seconds, with a final
44
extension step of 68˚C for 5 minutes. To assess the proper reverse transcription and
45
amplification of the sequences without interference of the tags, the amplification of the
46
positive strain of the viruses was realized in parallel. In the first step of the RT-PCR, only the
47
tagged forward primers (either tag-DWVrtF or tag-n6409-A1055R) were added and in the
48
second step, the tagged reverse primers (either tag- DWVrtR or tag-n6409-A1055R) were
49
added. After the RT-PCR cycles, amplicons were visualized by gel electrophoresis and
2
50
purified using USB ExoSAP-IT (Affymetrix), followed by Sanger sequencing on a capillary
51
sequencer at Massey Genome Service (Palmerston North, New Zealand).
52
53
Assessment of LHUV-1 and DWV infection rates
54
To gain an assessment of the prevalence of n6409 (LHUV-1) and DWV infections in
55
Argentine ants from Paraparaumu, near Wellington, New Zealand (40.917° S, 175.017° E).
56
We used 15 workers collected in Ambion RNAlater (Life Technologies) at the end of the
57
austral summer of March 2014. Additional queens and workers were collected in the austral
58
winter of June 2015. Ants were frozen alive and stored at -80°C. Samples collected in
59
summer were analysed for viral presence and active replication, as above. For samples in
60
collected in winter, RNA was extracted from 14 individual queens, and one pool of 30
61
workers, using a GeneJET Viral DNA & RNA Purification Kit (Thermo Scientific, New
62
Zealand) following the modified protocol previously described. The presence / absence of
63
DWV and LHUV-1 was confirmed following the previously described RT-PCR protocol. For
64
viral detection for the individual queen extractions, ~125 ng of RNA per sample was used.
65
Amplicons were visualized by gel electrophoresis, followed by purification using USB
66
ExoSAP-IT (Affymetrix) and Sanger sequencing on a capillary sequencer to Massey
67
University (Genome Service, Palmerston North, New Zealand). Sanger sequences were
68
examined in MEGA 6.06 and aligned with ClustalW (using default parameters) to their
69
original contig sequences to confirm the Sanger sequence identity.
70
Once the identity of the RT-PCR products were confirmed, replication of the viruses
71
was assessed using tagged primers and the modified one step RT-PCR previously described.
3
72
73
ESM Table 1. Tagged primers used to amplify the RNA negative strand of Deformed wing virus (DWV) and L. humile virus-1 (LHUV-1) using a
74
modified one step RT-PCR protocol. The tag sequence is in lower case.
Virus target
Primer name
Primer sense
Primer sequence 5’  3’
tag-DWVrtF
Forward
ttcgcagtaaGCAGCTGGAATGAATGCAGAGA
tag- DWVrtR
Reverse
ccagtaactcACGCGCTTAACACACGCAAA
tag-n6409-A656F
Forward
gctattgagtAGGAAACGCAGCCAGTGCTATT
tag-n6409-A1055R
Reverse
ttcttctatgGTCACCTGACTCCTTGCCTGATTT
DWV1
295
LHUV-1
75
1
Product length (bp)
399
modified from Lester et al. [3]
76
77
4
78
ESM Table 2. Published primers used to assess the presence / absence Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), Acute bee
79
paralysis virus (ABPV), Solenopsis invicta virus -1 and -2 (respectively SINV-1 and SINV-2), and Deformed wing virus (DWV) in Argentine ant
80
samples from Argentina, Australia and New Zealand. These viruses were chosen for analysis based on their previous observation in a range of
81
insect hosts, including ants [1,3,8,9,10].
RNA virus
IAPV, KBV, ABPV
[4]
Primer name
AKIF
AKIR
IAPV
[5]
ORF2-F8092
ORF2-R8318
IAPV-01F
IAPV-02R
KBV-F
KBV-R
IAPV
[6]
KBV
[7]
SINV-1
[8]
SINV-1
[8]
SINV-2
[9]
SINV-2
[9]
DWV
[3]
p117
p118
p114
p116
p64
p65
p548
p555
DWVrtF
DWVrtR
Primer orientation
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Primer sequence (5'  3')
CTTTCATGATGTGGAAACTCC
AAACTGAATAATACTGTGCGTA
CCAGCCGTGAAACATGTTCTTACC
ACATAGTTGCACGCCAATACGAGAAC
AAACATCACAGATGCTCAGGGTCGAGACTATATGT
CTAGGGAGCTACGGAGCGTGATTCGCCTTGTAGCT
GATGAACGTCGACCTATTGA
TGTGGGTTGGCTATGAGTCA
Reverse
Forward
Reverse
Forward
Reverse
Forward
CACTCCATACAACATTTGTAATAAAGATTTAATT
CCAATACTGAAACAACTGAGACACG
CTTGATCGGGCAGGACAAATTC
GAACGCTGATAACCAATGAGCC
ATTTGTTTGGCCACGGTCAAC
GATGATACAAAGCATTAGCGTAGGTAAACG
TGCATACTCGTTGTAAACAATCTGCTCATCT
Reverse
Forward
Reverse
TGCCGTGACAATCCTGAATATCGTCAGATGTA
GCAGCTGGAATGAATGCAGAGA
ACGCGCTTAACACACGCAAA
Product length (bp)
not clear in the article
226
427
393
154
647
318
717
295
82
83
5
84
ESM Table 3: Table of the 10 closest matches, ordered by the highest score for each contig of interest after BLASTN search on Genbank.
85
Abbreviations correspond to: Acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV) and Solenopsis
86
invicta virus-2 (SINV2).
87
Contig ID#
n1000
n1905
n6409 /
LHUV-1
ABPV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
ABPV
KBV
Max
score
300
291
285
285
279
273
273
273
273
273
910
Total
score
662
483
500
448
494
483
489
483
489
607
910
% query
coverage
44
43
43
42
43
43
43
43
43
42
97
Evalue
8e-77
6e-74
3e-72
3e-72
2e-70
1e-68
1e-68
1e-68
1e-68
1e-68
0.0
% query
identity
77
72
72
72
72
72
72
72
72
77
95
ABPV
825
825
94
0.0
93
HM228893
ABPV
825
825
94
0.0
93
HM228890
KBV
KBV
KBV
KBV
KBV
KBV
KBV
KBV
ABPV
737
735
690
683
681
681
675
81.4
64.1
737
735
690
683
681
681
675
81.4
64.1
97
71
64
74
71
71
71
7
7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
4e-11
6e-06
89
98
99
94
95
95
95
80
78
KF956377
AY787143
KC513761
AF177935
AF093457
AF034542
AF135854
AY275710
AY053368
Closest match name
Accession #
Taxa or description of match
AF150629
KC690268
KC690270
EU436455
KC690269
HQ897161
EU224280
EU218534
EU436423
AF486072
AY275710
Virus in honey bees (Apis mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus, origins not specified
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus used to infect honey bees (A. mellifera)
Virus in bat guano (Tadarida brasiliensis, Myotis
velifer, Nycticeus humeralis, Perimyotis subflavus)
Virus in bat guano (Tadarida brasiliensis, Myotis
velifer, Nycticeus humeralis, Perimyotis subflavus)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in mites (Varroa jacobsoni)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus used to infect honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
6
n1050
ABPV
ABPV
IAPV
64.1
64.1
62.2
64.1
64.1
62.2
7
7
6
6e-06
6e-06
2e-05
78
78
78
AY053367
AY053366
JX045857
ABPV
62.2
62.2
6
2e-05
78
HM228893
ABPV
62.2
62.2
6
2e-05
78
HM228890
IAPV
IAPV
IAPV
SINV-2
Acyrthosiphon pisum
Carica papaya
Shewanella loihica
Oryzias latipes
Hymenolepis
diminuta
Cyprinus carpio
Dictyostelium
discoideum
Ceratitis capitata
Staphylococcus
aureus
62.2
62.2
62.2
56.4
50.7
48.8
48.8
46.8
62.2
62.2
62.2
56.4
50.7
48.8
48.8
46.8
6
6
6
6
1
1
1
1
2e-05
2e-05
2e-05
0.002
0.096
0.36
0.36
1.4
78
78
78
74
94
96
94
100
46.8
46.8
1
1.4
96
EU436455
EU436528
EU436527
EF428566
XM_001943802
KM397499
CP000606
HG314000
LM385892
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in bat guano (Tadarida brasiliensis, Myotis
velifer, Nycticeus humeralis, Perimyotis subflavus)
Virus in bat guano (Tadarida brasiliensis, Myotis
velifer, Nycticeus humeralis, Perimyotis subflavus)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in honey bees (A. mellifera)
Virus in fire ants (Solenopsis invicta)
Aphids mRNA
Papayas mRNA
Bacteria genome
Fish genome
Tapeworms genome
46.8
46.8
1
1.4
91
46.8
46.8
1
1.4
96
LN594801
XM_629989
Common carp genome
Amoeba mRNA
44.9
44.9
1
5.2
93
44.9
44.9
1
5.2
96
XM_004522381
CP009554.1
Fruit flies mRNA
Bacteria genome
88
89
7
90
ESM Table 4: Table of the 10 closest matches, ordered by the highest score for each contig of interest after BLASTX search on Genbank.
91
Abbreviations correspond to: Acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV) and Solenopsis
92
invicta virus-2 (SINV2).
93
Contig ID#
n1000
n1905
n6409
Closest match name
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
KBV
KBV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
KBV
IAPV
IAPV
KBV
Max
score
571
571
569
568
566
565
565
565
565
565
388
383
382
380
380
379
379
379
379
393
383
384
380
Total
score
571
571
569
568
566
565
565
565
565
565
388
383
382
380
380
379
379
379
379
393
383
384
380
% query
coverage
41
41
41
41
41
41
41
41
41
41
99
99
99
99
99
99
99
99
99
93
95
95
90
E-value
7e-177
9e-177
4e-176
7e-176
3e-175
8e-175
8e-175
9e-175
1e-174
1e-174
4e-121
2e-119
4e-119
4e-118
4e-118
5e-118
5e-118
5e-118
6e-118
1e-123
3e-121
3e-120
4e-120
% query
identity
76
76
76
76
76
76
76
76
76
75
96
94
94
94
94
94
93
93
93
46
44
44
46
Accession #
ACD01399
ABY71756
ACD01403
ABY71754
ABY57949
ACD01401
AGL33503
AEL12438
AGL33499
AGF84784
NP_851403
AHL83499
ACD01403
ACD01401
ABY57949
YP_001040002
AGL33499
AEL12438
ACD01399
NP_851404
AGF84785
ACD01404
AAR19088
Taxa or description of match
Virus polyprotein
Virus non-structural polyprotein
Virus polymerase polyprotein
Virus non-structural polyprotein
Virus non-structural polyprotein
Virus polymerase polyprotein
Virus polymerase polyprotein
Virus polymerase polyprotein
Virus polymerase polyprotein
Virus non-structural polyprotein
Virus non-structural polyprotein
Virus non-structural polyprotein
Virus polymerase polyprotein
Virus polymerase polyprotein
Virus non-structural polyprotein
Virus polymerase polyprotein
Virus polymerase polyprotein
Virus polymerase polyprotein
Virus polymerase polyprotein
Virus structural polyprotein
Virus structural polyprotein
Virus structural polyprotein
Virus structural polyprotein
8
n1050
IAPV
IAPV
IAPV
IAPV
IAPV
IAPV
SINV2
Bovine kobuvirus
Salivirus
Caprine kobuvirus
Porcine kobuvirus
swine
Bovine kobuvirus
Kobuvirus sheep
Porcine kobuvirus
Kobuvirus
Carrot necrotic
dieback virus
384
383
383
382
382
382
452
84.0
81.3
80.9
80.5
384
383
383
382
382
382
452
84.0
81.3
80.9
80.5
95
95
95
95
95
95
72
21
23
21
21
6e-120
1e-119
2e-119
2e-119
3e-119
3e-119
1e-136
4e-14
3e-13
4e-13
5e-13
44
44
44
44
44
44
50
34
32
31
33
ABY57950
ACD01400
ABY71757
AGL33500
AEL12439
ACD01402
YP_001285729
NP_859024
YP_003038640
YP_009001376
YP_002473940
82.8
82.8
82.4
82.4
82.4
82.8
82.8
82.4
82.4
82.4
20
20
26
28
37
7e-13
8e-13
9e-13
9e-13
1e-12
35
35
31
28
29
NP_740257
ADG03747
AHY02128
AIK67137
ACJ04421
Virus structural polyprotein
Virus structural polyprotein
Virus structural polyprotein
Virus structural polyprotein
Virus structural polyprotein
Virus structural polyprotein
Virus non-structural protein
Virus non-structural protein 2C
Virus non-structural protein 2C
Virus non-structural protein 2C
Virus non-structural protein 2C
Virus hypothetical protein
Virus polyprotein
Virus polyprotein
Virus polyprotein
Virus polyprotein
94
9
95
96
97
98
99
ESM Figure 1. Maximum likelihood tree of Linepithema humile virus -1 (LHUV-1) protein
100
sequences (560 amino acids) with 1000 bootstraps of the WAG model [11] with uniform rates
101
in MEGA 6.06 [12]. The original LHUV-1 sequence (contig n6409) from the RNA
102
metagenome analysis is in bold red. Sequences obtained from one step RT-PCRs in this study
103
are in bold black. Sequences with * were among the 100 best hits of LHUV-1 using a
104
BLASTX search on GenBank and corresponded to two dicistroviruses, Kashmir bee virus
105
(KBV) and Israeli acute paralysis virus (IAPV). Country of origin (New Zealand – NZ,
106
Canada, and United States – USA) and host organisms (Argentine ants – L. humile, and honey
107
bees – Apis mellifera) are indicated for each sequence
108
10
109
110
111
ESM Figure 2. Maximum likelihood tree of RNA metagenome contig n1000 nucleotide
112
sequences with 1000 bootstraps of the Tamura-3 parameter model with uniform rates [13] in
113
MEGA 6.06. The original RNA metagenome contig n1000 from the preliminary
114
metagenomics analysis is in bold red. Sequences obtained from one step RT-PCRs in this
115
study are in bold black. Sequences with * were among the 100 best hits of contig n1000 using
116
a BLASTX search of GenBank and corresponded to the dicistrovirus Israeli acute paralysis
117
virus (IAPV). Country of origin (New Zealand – NZ, Australia, and Korea) and host
118
organisms (Argentine ants – L. humile, and honey bees – Apis mellifera) are indicated for
119
each sequence.
120
11
121
122
123
124
ESM Figure 3. Maximum likelihood tree of RNA metagenome contig n1050 nucleotide
125
sequences with 1000 bootstraps of the Tamura-3 parameter model with uniform rates (T92) in
126
MEGA 6.06. The original RNA metagenome contig n1050 from the preliminary
127
metagenomics analysis is in bold red. Sequences obtained from one step RT-PCRs in this
128
study are in bold black. Sequence with * was among the 100 best hits of contig n1050 from
129
BLASTX searches on GenBank, and corresponded to the dicistrovirus Solenopsis invicta
130
virus 2 (SINV-2). Country of origin (New Zealand – NZ, Australia, and United States – USA)
131
and host organisms (Argentine ants – L. humile, and red imported fire ants – S. invicta) are
132
indicated for each sequence.
133
12
134
135
ESM Figure 4. Maximum likelihood tree of RNA metagenome contig n1905 nucleotide
136
sequences with 1000 bootstraps of the Tamura-3 parameter model with uniform rates (T92) in
137
MEGA 6.06. The original RNA metagenome contig n1905 from the preliminary
138
metagenomics analysis is in bold red. Sequences obtained from one step RT-PCRs in this
139
study are in bold black. Sequences with * were among the best hits of contig n1905 from
140
BLASTX searches on GenBank and corresponded to the dicistrovirus Acute bee paralysis
141
virus (ABPV). Country of origin (New Zealand – NZ, United Kingdom– UK) and host
142
organisms (Argentine ants – L. humile, and honey bees – Apis mellifera) are indicated for
143
each sequence.
144
145
13
146
ESM References
147
148
149
150
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