AEM Accepted Manuscript Posted Online 6 July 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.01239-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Novel detection of low level Cardinium and Wolbachia infections in Culicoides
2
Running title: Low level endosymbionts in Culicoides
3 4
Peter T Mee1,2, Andrew R Weeks1, Peter J Walker2, Ary A Hoffmann1, Jean-Bernard
5
Duchemin2#
6
Keywords: Culicoides; endosymbionts; biting midge; Cardinium; Wolbachia
7 8 9 10
1
Bio21 Institute and School of BioSciences, The University of Melbourne, 3052,
Parkville, Australia. 2
CSIRO Biosecurity, Australian Animal Health Laboratory, Geelong, 3219, Victoria,
Australia.
11 12 13 14 15 16
Corresponding author
17
Jean-Bernard Duchemin [email protected]
18 19 20 21 22 23 24 1
25
Abstract
26
Bacterial endosymbionts have been identified as potentially useful biological control
27
agents for a range of invertebrate vectors of disease. Previous studies of Culicoides
28
(Diptera: Ceratopogonidae) species using conventional PCR assays have provided
29
evidence of Wolbachia (1/33) and Cardinium (8/33) infections. Here, we screened 20
30
species of Culicoides, for Wolbachia and Cardinium, utilizing a combination of
31
conventional PCR and more sensitive qPCR assays. Low levels of Cardinium DNA were
32
detected in females of all but one of the Culicoides species screened and low levels of
33
Wolbachia were detected in females of 9 of the 20 Culicoides species. Sequence
34
analysis based on partial 16S rDNA and gryB identified Candidatus Cardinium hertigii
35
from group C, which has previously been identified in Culicoides from Japan, Israel and
36
the United Kingdom. Wolbachia strains detected in this study showed 98-99% sequence
37
identity to Wolbachia previously detected from Culicoides based on 16S rDNA, whereas
38
a strain with a novel wsp sequence was identified in C. narrabeenensis. Cardinium
39
isolates grouped to geographical regions independent of host Culicoides species,
40
suggesting possible geographical barriers to Cardinium movement. Screening also
41
identified the bacteria Asaia in Culicoides. These findings point to a diversity of low level
42
endosymbiont infections in Culicoides, providing candidates for further characterization
43
and highlighting the widespread occurrence of these endosymbionts in this insect group.
44 45 46 47 48 2
49
INTRODUCTION
50
Culicoides are small haematophagous insects (Diptera: Ceratopogonidae), some of
51
which are important vectors of viral and parasitic diseases of veterinary and medical
52
importance (1). More than 50 different viruses have been isolated from Culicoides
53
species, including bluetongue virus (BTV), Schmallenberg virus (SBV) and African
54
horse sickness virus (AHSV) which cause significant impacts on livestock production
55
through stock losses and trade restrictions (1). Capable of wind-borne displacement for
56
several hundred kilometers, Culicoides have a capacity for rapid long distance
57
transmission of disease and have recently been responsible for the establishment of
58
enzootic BTV and SBV infections over vast new geographic areas (1, 2). Current control
59
methods for Culicoides include breeding site removal, baiting of livestock and midge
60
resting sites; however these techniques are costly, labor intensive and have varying
61
levels of success and permanence of control (3). Although vaccines are available for
62
some Culicoides-transmitted viruses, such as BTV, the practicality of vaccination is
63
limited by the large number of BTV serotypes and the potential for genome segment
64
reassortment between live vaccines and naturally circulating virus strains (4).
65
Inactivated vaccines are expensive and less potent but have been used effectively in
66
Europe for control of BTV (5). Inactivated vaccines are not currently available for AHSV.
67
Insect vector control by use of endosymbiotic organisms has gained increasing
68
attention in recent years (6). Bacterial endosymbionts present in arthropod species are
69
capable of influencing host characteristics such as longevity and vector competence, as
70
well as being involved in nutrient provisioning (7-9). The endosymbiont Wolbachia
71
pipientis (Alphaproteobacteria) has attracted notable attention for its applicability to
72
endosymbiont-based control of the dengue virus vector, Aedes aegypti. Wolbachia has 3
73
been shown to successfully invade and be maintained in natural A. aegypti populations
74
and to block virus transmission (8, 10-12).
75
Several previous studies have reported evidence of bacterial endosymbiont
76
infection in Culicoides species. Screening studies conducted using conventional PCR
77
assays detected Wolbachia DNA in a single Culicoides paraflavescens individual in
78
Japan
79
endosymbiont which also has a range of influences on its host insect, has been
80
detected in four Culicoides species in Japan, two Culicoides species in Israel and two in
81
the United Kingdom (13-15). Endosymbiotic diversity in Australian Culicoides species
82
has not been investigated previously, nor has a comparative analysis of Cardinium
83
divergence in different Culicoides species from diverse geographical locations been
84
reported.
(13).
Candidatus
Cardinium
hertigii
(Bacteroidetes),
another
bacterial
85
Although conventional PCR has previously been used successfully to screen
86
arthropods for endosymbionts (16-18), recent studies have demonstrated that this
87
method can fail to detect low level infections. More sensitive screening techniques such
88
as long-(19), nested-(20) or quantitative-PCR (21) are therefore required. Low level
89
endosymbiont infections have been identified in a range of insects from tsetse flies (22),
90
Drosophila (23), Cherry fruit fly (24) and planthoppers (25). Previous studies have
91
suggested that at low levels of infection endosymbionts are capable of influencing the
92
host. For example, low levels of Wolbachia in Drosophila paulistorum semispecies have
93
been shown to influence fecundity, sex ratios and mate discrimination (23). However
94
other endosymbiont effects including viral blockage and fitness effects may depend on
95
bacterial density (26).
4
96
In this study, a range of Culicoides species, collected predominately from south-
97
eastern Australia, were screened for evidence of Cardinium and Wolbachia infection.
98
Global movement of Cardinium in Culicoides species was also investigated based on
99
sequence divergence in multiple loci. Novel Cardinium and Wolbachia infections were
100
identified in a range of Culicoides species, a high proportion of which were low-level
101
infections. Nucleotide sequence analysis revealed that Cardinium detected in these
102
samples was genetically similar to those previously discovered in Japan, Israel and the
103
United Kingdom, suggesting a global presence of a single Cardinium strain throughout a
104
wide geographical range and in a range of Culicoides species.
105 106
MATERIALS AND METHODS
107 108
Insect collection. Culicoides were collected using either Centre for Disease Control
109
(CDC) mini light traps (BioQuip Products, Rancho Dominguez, CA) or yellow sticky
110
traps (YST). Mini light traps contained either green or ultraviolet LEDs and were
111
powered by a 6 V motorbike battery (27, 28). Traps were positioned approximately 2 m
112
above the ground at dusk and collected after dawn, consistent with previously trapping
113
methodologies (29). A downdraught fan in the traps directed insects into a beaker
114
containing approximately 200 ml of 80% ethanol. Contents of the beakers were
115
collected daily. Before storing at -20 °C, insects were sorted, identified and stored
116
individually in fresh 80% ethanol. By washing insects in ethanol, the risk of
117
contamination was reduced. Yellow sticky traps (YST) (Trappit) were elevated
118
approximately 10 cm above the ground by a plastic stick and illuminated by a solar
119
garden light. Insects were individually cut from the YST and placed in a tube containing 5
120
enough solvent (De-Solv-it®, orange oil) (Orange-Sol, Chandler, AZ) to cover the
121
sample. Tubes were incubated at 60 oC for 5 min with intermittent inversion until the
122
insect floated off the YST. Insects were removed and washed twice with 80% ethanol
123
before storing in fresh 80% ethanol at -20 oC (I. Valenzuela pers. comm.). Collections
124
were made from 305 traps (81 CDC & 224 YST) over a period of 33 trapping nights
125
throughout south-eastern Australia from January to April in 2013 and 2014. Samples of
126
C. imicola collected using CDC light traps were also obtained from Kenya and
127
Madagascar, through the International Livestock Research Institute, Kenya. Culicoides
128
imicola were identified morphologically based on wing patterning, and through sequence
129
analysis of the cytochrome oxidase subunit 1 (COI) region.
130 131
Culicoides identification. Morphological identification was performed with the aid of
132
the pictorial atlas of Australasian Culicoides wings (Diptera: Ceratopogonidae) (30). The
133
C. victoriae species group was separated into a series of species denoted by wing
134
identifier number in square brackets, such as C. victoriae [241], based on the wing
135
pictures from Dyce et al. (2007). One C. victoriae species not present in pictorial atlas
136
was collected in Geelong, Victoria and is denoted as C. victoriae [172] (BOLD sequence
137
ID CUVIC12-15). Genetic confirmation of Culicoides species was based on amplification
138
and sequencing of the COI, which is commonly used for species determination of
139
Culicoides and capable of differentiating most Culicoides species (31, 32).
140 141
DNA extractions. Culicoides were removed from ethanol and air dried before extraction
142
(33). The mouse tail protocol of the MagMaxTM-96 DNA Multi-Sample Kit (Applied
143
Biosystems, Austin, TX) on a KingFisherTM Flex (Thermo Scientific, Waltham, MA) was 6
144
used with the following amendments. Individual Culicoides were incubated in 100 µl of
145
PK buffer and 10 mg/ml of Proteinase K. Samples were homogenized with a
146
FastPrep®-24 Instrument (MP Biomedicals, Santa Ana, CA) at 6.0 m/s for 20 s with
147
approximately 10 x 1.0 mm zirconia/silica beads (BioSpec Products, Bartlesville, OK)
148
before overnight incubation at 56 oC. Elution was altered to 40 µl of both elution buffer 1
149
and 2. To assess possible contamination during extraction every eleventh well in the
150
MagMaxTM 96-well plate was left without a Culicoides sample, but was still included in
151
all downstream screening steps.
152 153
Conventional PCR screening. Primers (GeneWorks, SA, Australia) utilized included
154
an initial housekeeper targeting the third loop of the 28S ribosomal DNA (rDNA) (D3a,
155
D3ba),
156
Alphaproteobacteria 16S rDNA (Alf28F, Alf684R) (34, 35), Wolbachia 16S rDNA (Wol-F,
157
Wol-R) (36) and cytochrome oxidase subunit 1 (COI) (BC1culicFm, JerR2m) (32), for
158
species confirmation. Primer sequences can be found in Table 1. 25 µL PCR reactions
159
were performed using 1 unit Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad,
160
CA), following manufacturer’s protocol. PCR conditions were as follows: 1 cycle 95 oC
161
for 2 mins; 35 cycles of 95 oC for 30 sec, anneal temperature as in Table 1 for 30 sec,
162
72 oC for 30 sec; and final extension at 72 oC for 5 min. Two negative controls of ultra
163
pure water were included for all PCR screens to ensure no contamination occurred. A
164
single positive control was included for each PCR, DNA from C. victoriae [240] was
165
used for 28S and COI PCR, and DNA from Drosophila simulans was used for
166
Wolbachia 16S and Alphaproteobacteria 16S PCR. At the commencement of screening
167
no positive control was available for Cardinium; in later steps C. victoriae [240] samples
followed
by
Cardinium
16S
rDNA
(CAR-SP-F,
CAR-SP-R)
(13),
7
168
shown to be Cardinium-positive were used as a control. Amplified products were
169
separated on a 1% agarose gel containing 0.1% SYBR® Safe DNA Gel Stain
170
(Invitrogen, USA) and visualized with a G:BOX Syngene blue light visualization
171
instrument.
172 173
Multi-allele typing. Samples which tested positive for Cardinium by conventional PCR
174
screening were typed for multiple alleles using 16S rDNA and DNA gyrase subunit B
175
(gyrB). Four primers (16SA1F, 16SA1R, 16SA2F & 16SA2R) (Table 2) were designed
176
to amplify two overlapping regions of Cardinium 16S rDNA, based on the Clustal W
177
alignment of sequences from Culicoides (AB506776-AB506779, JN166961, JN166962)
178
available in the GenBank database. Primers were designed using Geneious v7.0.5, and
179
covered 1,404 bp of the 16S rDNA gene, with 243 bp overlap between the amplicons
180
(37). Four primers (gyrBA1F, gyrBA1R, gyrBA2F & gyrBA2R) targeting a 1,399 bp
181
region of gyrB gene with 168 bp overlap between amplicons (Table 2) were designed
182
using the same process as above, with sequences available in GenBank (AB506791,
183
AB506792, JN166963, JN166964). PCR reactions were performed using the conditions
184
as previously stated, an annealing temperature of 50 °C was used for Cardinium 16S
185
rDNA and gyrB primers.
186
Low level Wolbachia detections were amplified using a combination of hemi-
187
nested (16S) and nested (wsp) PCRs. Hemi-nested primers for 16S used two sets of
188
previously published primers from Wol-F and Wol-R (36) and Rao-F and Rao-R (38).
189
First round amplification was performed using the Wol-F and Wol-R as previously
190
outlined, generating an 897 bp amplicon. Second round amplification yielded two
191
amplicons, amplicon 1 = 464 bp (Wol-F and Rao-R) and amplicon 2 = 495 bp (Rao-F 8
192
and Wol-R); both rounds of amplification had an annealing temperature of 52 oC. A
193
nested-PCR developed by Hughes et al. (2012) (Table 1) was used to amplify a 423 bp
194
region of the wsp. The nested protocol was altered to a two-step nested PCR instead of
195
the previously outlined single tube nested-PCR (39). Two negative controls both
196
involving ultra-pure water as template were run for first round amplification, with the
197
product used as template for second round amplification.
198 199
Quantitative PCR screening. Quantitative PCR (qPCR) was used to increase the level
200
of sensitivity of detection of Wolbachia and Cardinium. Samples screened with the
201
conventional assays were rescreened with the qPCR assays. A Wolbachia qPCR was
202
obtained based on the primer and probe design of Rao et al. (2006), which used this
203
assay to detect Wolbachia in field collected mosquitoes. The probe reporter dye at the
204
5’ end was changed to VIC (4, 7, 2’-trichloro-7’-phenyl-6-carboxyfluorescein) as
205
opposed to FAM (6-carboxyfluorescein) but retained the same TAMRA (6-
206
carboxytetramethylrhodamine) quencher dye at the 3’ end. Wolbachia primers and the
207
probe reaction setup was modified to a 20 µl reaction using 10 µl of TaqMan® Universal
208
PCR Master Mix (Applied Biosystems, Foster City, CA), 1 µl of both primers at 0.3 µM
209
forward, 0.9 µM reverse, 1 µl of 0.05 µM probe, 2 µl of template DNA and 5 µl of ultra-
210
pure water.
211
To develop a Cardinium qPCR, published Cardinium sequences isolated from
212
Culicoides (AB506776-AB506779, JN166961 and JN166962) were aligned using
213
Geneious v7.0.5 and a consensus sequence obtained. Primer Express® software
214
v3.0.1. (Applied Biosystems) was used to design a TaqMan probe and primer pair to
215
amplify a 111 bp region (Table 3) to specifically target Cardinium group C. Cardinium 9
216
primers and probes were calibrated to a final reaction volume of 20 µl consisting of 10 µl
217
of TaqMan® Universal PCR Master Mix, 2 µl of 0.3 µM forward and reverse primer, 2 µl
218
of 0.05 µM probe, 2 µl of template DNA and ultra-pure water. Optimal primer and probe
219
concentrations for both assays were determined as in the protocol for the TaqMan®
220
Universal PCR Master Mix. Triplicate reactions were performed on a QuantStudioTM 6
221
real-time PCR machine (Life Technologies, Carlsbad, CA). Cycling conditions included
222
an initial incubation at 50 oC for 2 min to activate uracil-N-glycosylase, followed by
223
denaturation 10 min at 95 oC then 45 cycles of 95 oC for 15 s and a combined annealing
224
and primer extension phase at 60 oC for 1 min. Screening was conducted in a 96 well
225
plate format with 6 wells each plate being left for use as negative controls.
226
Cloned standards of Cardinium and Wolbachia were generated and used as
227
positive controls, as follows. Conventional PCR was conducted using qPCR primers to
228
amplify known Wolbachia positive material from Drosophila simulans and Cardinium
229
positive material from Culicoides victoriae [240]. Amplicons were visualized on a 1%
230
agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA).
231
Purified DNA was ligated into pGEM®-T Easy Vector (Promega, Madison, WI)
232
according to the manufacturer’s protocol, then electroporated into competent E. coli
233
DH5α cells (New England Biolabs, Beverly, MA). Plasmid DNA was purified from
234
positive clones using QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA) and sequenced
235
using SP6 and T7 sequencing primers. Plasmid DNA clones were serially diluted before
236
being used as positive controls. Wolbachia and Cardinium plasmids was also used to
237
generate a standard regression curve based on 6 serial dilutions of the plasmid in each
238
qPCR run, allowing relative quantification of infection. Plasmid copy numbers were
10
239
calculated
as
per
established
240
(http://www6.appliedbiosystems.com/support/tutorials) (Applied Biosystems).
protocols
241 242
Sensitivity comparison of conventional and qPCR. Detection limits of conventional
243
and quantitative PCR were assessed by serially diluting Wolbachia positive template
244
DNA from Drosophila simulans and Cardinium positive template DNA from Culicoides
245
victoriae [240]. Ultra-pure water was used to make 10-fold serial dilutions of each DNA
246
template from neat to 10-9. Duplicate 2 µl samples of each dilution were tested using the
247
Cardinium conventional (CAR-SP-F, CAR-SP-R) (13) and quantitative assays (Table 3),
248
as well as the Wolbachia conventional (Wol-F, Wol-R) (36) and quantitative assays
249
(Table 3).
250 251
Sequencing. Nucleotide sequences of Cardinium, Wolbachia and Alphaproteobacteria
252
positive amplicons were determined for species confirmation. DNA was purified from
253
positive amplicons with a QIAquick Gel Extraction Kit (Qiagen, Santa Clara, CA)
254
according to the manufacturer’s protocol. Fluorometric quantification of purified DNA
255
was performed using a dsDNA HS Assay on a Qubit® (Invitrogen, Carlsbad, CA).
256
Sequencing was performed using the BigDye® Terminator v3.1 kit (Applied Biosystems,
257
Foster City, CA) according to the manufacturer’s protocol and sequence was generated
258
via capillary Sanger sequencing on a 3500 Genetic Analyzer (Applied Biosystems).
259
A series of measures was implemented as an added precaution to reduce the
260
risk of contamination and to ensure no false-positive detections. Clean-room working
261
conditions were utilized with insect identification, nucleotide extraction, master mix set
11
262
up, amplification and post-amplification handling all being performed in separate labs,
263
with a non-amplified to amplified workflow being followed.
264
To compare the incidence of infections across sexes, contingency analyses were
265
carried out for species where the infection was detected in at least one sex. Probabilities
266
for detecting associations between infections and sex were based on exact tests.
267 268
Phylogenetic analysis. Phylogenetic relationships of Cardinium were analyzed using
269
nucleotide sequences obtained from 16S rDNA and gyrB genes. Additional group A
270
(isolated from Ixodes scapularis (AB001518, AB506790), Euides speciosa (AB506775,
271
AB506788), Tetranychus pueraricola (AB241135, AB506784), Oligonychus ilicis
272
(AB241130, AB506783)) and group B (isolated from Paenicardinium endonii
273
(DQ314214, DQ314215)) Cardinium sequences obtained from GenBank were included
274
to provide phylogenetic resolution. Cardinium 16s rDNA and gyrB sequences were
275
concatenated head-to-tail forming a super-gene alignment. Concatenating two genes
276
together increases the number of evolutionary changes on individual branches which in
277
turn can generate a more precise phylogeny, as opposed to when the two genes are
278
analyzed separately (40). The concatenated alignment was screened for the presence
279
of recombination using the RDP, GENECONV, Bootscan, MaxChi, Chimaera and
280
SiScan methods implemented in Recombination Detection Program v4.39 (RDP4), with
281
default parameters (41). A Wolbachia phylogeny was determined using 16S rDNA and
282
Wolbachia Surface Protein (wsp). The wsp gene was included in phylogenetic analysis
283
as it has been shown to have a much greater divergence rate than 16S rDNA (18, 36).
284
Wolbachia sequences from supergroup A (Drosophila sechellia), B (Culex pipiens) and
285
C (Dirofilaria immitis) were included to see where Culicoides Wolbachia would be 12
286
placed within the wider Wolbachia phylogeny, supergroup C being used to root the
287
trees. Host Culicoides phylogenetic analysis was based on mitochondrial cytochrome
288
oxidase subunit I (COI).
289
All sequences were aligned with the Clustal W algorithm in Geneious v7.0.5
290
(Biomatters Ltd) (37). Aligned nucleotide sequences were analyzed using jModelTest2
291
v2.1.3, with the topology search taking the best of Nearest Neighbor Interchange (NNI)
292
and Subtree Pruning and Regrafting (SPR) (42). Evolutionary models implemented to
293
construct phylogenetic trees were selected based on the Akaike Information Criterion
294
output from JModelTest2 (42) (number of substitution schemes 11, base tree for
295
likelihood calculations Maximum-Likelihood (ML) optimized, base tree search best). The
296
Hasegawa-Kishino-Yano (HKY) model was selected for Cardinium 16S rDNA,
297
Wolbachia wsp and the concatenation of Cardinium 16S rDNA and gyrB. The General
298
Time-Reversible (GTR) model was selected for the Cardinium gyrB, Wolbachia 16S
299
rDNA and Culicoides COI phylogeny. Phylogenetic ML trees were constructed using the
300
PhyML plugin in Geneious v7.0.5 with 1,000 bootstrap replicates; the proportion of
301
invariable sites and gamma distribution were both estimated (43).
302 303
Nucleotide sequence accession numbers. Sequences generated were deposited in
304
GenBank. Cardinium accession numbers for 16S rRNA are as follows KR026906-
305
KR0269018, KR0269020-KR026923, and gyrB gene are KR026924-KR026935.
306
Wolbachia accession numbers for 16S rRNA are KR026936-KR026941 and wsp are
307
KR026942-KR026951. Sequences for the Culicoides victoriae group were deposited in
308
Barcode of Life Data Systems (BOLD) database under the sample ID’s CUVIC10-15 to
309
CUVIC18-15. 13
310 311
Mapping of Cardinium and Wolbachia in Culicoides. Culicoides which tested
312
positive for Wolbachia or Cardinium based on screening with the quantitative assays,
313
were plotted against capture location. Shapefiles were obtained from the Australian
314
Bureau of Statistics, Australian Standard Geographical Classification (ASGC) Digital
315
Boundaries, Australia July 2011. The distribution map was created in quantum GIS
316
(QGIS) Wien v2.8.1 (44).
317 318
RESULTS
319 320
Endosymbiont assays. Increased sensitivity for the Wolbachia and Cardinium
321
quantitative PCRs was obtained in comparison to the conventional assays. Serial
322
dilution of Wolbachia and Cardinium positive samples saw a 100-fold lower detection
323
limit by the qPCR assays. Detections were confirmed by sequencing, however the
324
Wolbachia (62 bp of 16S) and Cardinium (111 bp of 16S) qPCR amplicons were too
325
small for species discrimination. Developed nested-PCRs for Wolbachia (16S and wsp)
326
and Cardinium (16S and gyrB) were used to amplifying enough material of the level low
327
detections to allow confirmation by sequencing.
328
Twenty Culicoides species were identified by morphological and genetic typing of
329
insects collected in Victoria, Tasmania and Queensland from January to April 2013 and
330
2014 (Fig. 1). These samples, together with samples of C. imicola from Kenya and
331
Madagascar, were analyzed for evidence of Cardinium and Wolbachia infection through
332
the conventional and quantitative PCR assays.
333 14
334
Cardinium screening. The conventional PCR assay identified evidence of Cardinium
335
infection in females of three species, C. victoriae [240] and C. brevitarsis from Australia,
336
and C. imicola from Kenya (Table 4). Rescreening the samples using the qPCR assay
337
indicated a substantially higher prevalence of Cardinium infection, with positive samples
338
detected in all Culicoides species except for C. victoriae [172] (Table 4). The percentage
339
of positives detected in females increased from 4% (14/360, CI: 2.23 - 6.28%)
340
(conventional PCR) to 26% (92/360, CI: 21.25 - 30.25%) (qPCR), and detections in
341
males increased from 0% (0/161, CI: 0 - 1.84%) (conventional PCR) to 14% (22/161, CI:
342
8.98-19.64%) (qPCR). A lower number of male Culicoides were screened due to their
343
lower representation in collections. However, in those Culicoides species in which
344
Cardinium was detected in individuals of both sexes, there was no significant difference
345
between prevalence in males and females for C. brevitarsis (contingency analysis, P =
346
0.63, df = 1), C. bundyensis (contingency analysis, P = 0.33, df = 1), and C. victoriae
347
[245] (contingency analysis, P = 0.24, df = 1), although differences were marginally non-
348
significant in the case of C. parvimaculatus (contingency analysis, P = 0.065, df = 1) and
349
C. austropalpalis (contingency analysis, P = 0.067, df = 1) due to a higher prevalence in
350
female Culicoides . For C. marksi no infection was identified in males but it was present
351
in some females (contingency analysis, P = 0.035).
352
Detections of Cardinium by conventional PCR were seen at the lowest Cq values of
353
26.58 in C. williwilli (equating to 23,000,000 copies) and the highest Cq value of 34.06
354
(equating to 24,000 copies) in C. victoriae [240] (Table S1). However, the Cardinium
355
qPCR assay provided detections at the lowest Cq values of 42.68 in C. parvimaculatus
356
which equates to approximately 290 copies of the Cardinium target region. In Culicoides
357
species which had detections of Cardinium in both sexes, there was no significant 15
358
difference (based on t-tests) in Cq values and hence Cardinium copy number between
359
sexes in C. bundyensis (P = 0.22, df = 10) and C. austropalpalis (P = 0.29, df = 8),
360
although there was a difference for C. parvimaculatus (P = 0.018, df = 8).
361 362
Wolbachia screening. No evidence of Wolbachia infection was detected in any of the
363
Culicoides samples tested using the conventional PCR. In contrast, evidence of low-
364
level Wolbachia infections was detected in ten species of Culicoides when individual
365
samples were screened using the qPCR assay (Table 4). Across all species tested, the
366
percentages of detected positive females (7%, 23/353, CI = 4.27 – 9.46) and males
367
(5%, 8/160, CI = 2.34 – 9.27) were similar (contingency test, P = 0.55, df = 1).
368
Culicoides bundyensis was the only species in which evidence of Wolbachia infection
369
was detected in both sexes, with a similar proportion of positive males and females
370
(contingency test, P = 0.54, df = 1).
371
Detections of Wolbachia by qPCR were seen to range from the lowest Cq value
372
of 30.53 in C. antennalis (equating to 11,600 copies) and the highest Cq value of 41.30
373
(equating to 22.4 copies) in C. brevitarsis (Table S1). Detections of Wolbachia were
374
rarely seen in both sexes of the same Culicoides species, hence no comparison of Cq
375
value and sex were performed.
376
There was evidence of low-level dual infections with Wolbachia and Cardinium in
377
a number of Culicoides species. These included five individuals of C. antennalis, four
378
individuals of C. brevitarsis and C. parvimaculatus, three individuals of C. bundyensis
379
and C. victoriae [245], and single individuals of C. imicola (Madagascar), C. marksi, C.
380
austropalpalis and C. marmoratus (Table 5). The dual infections in single individuals
16
381
occurred at the expected frequency on the basis of a random association between the
382
infections.
383 384
Alphaproteobacteria screening. Screening by conventional PCR was also performed
385
using the broad range Alphaproteobacteria primer set. The assay provided evidence of
386
infection in individuals representing four Culicoides species: C. austropalpalis in which
387
30% (13/43, CI = 17.96 – 45.09%) females and 11% (3/28, CI = 2.79 – 26.45%) males
388
were positive; C. marksi in which 7% (2/29, CI = 1.17 – 20.97%) females and 5% (1/19,
389
CI = 0.26 – 23.33%) of males were positive; C. brevitarsis in which 22% (2/9, CI = 3.90
390
– 56.21%) females and 50% (1/2, CI = 2.5 – 97.5%) males were positive; and C.
391
bundyensis in which 18% (5/27, CI = 7.11 – 36.38%) females were positive. The
392
amplicons generated in the assay were sequenced. BLAST analysis indicated that
393
amplicons obtained from C. marksi and C. brevitarsis shared a high percentage of
394
nucleotide sequence identity (99%) with the 16S ribosomal DNA gene of an Asaia sp.
395
bacterium isolated from Aedes albopictus mosquitoes (Genbank JX445140.1).
396 397
Cardinium phylogenetic analysis. Partial 16S ribosomal DNA and DNA gyrase
398
subunit B gene sequences were used to construct the Cardinium phylogeny. Based on
399
the 505 bp 16S rDNA amplicon, all Australian Cardinium isolates grouped with the
400
previously described sequences of Culicoides group C Cardinium hertigii from Israel (C.
401
oxystoma and C. imicola), Japan (C. arakawae, C. lungchiensis, C. peregrinus and C.
402
ohmorii) and the United Kingdom (C. punctatus and C. pulicaris) (Fig. S1a). A low level
403
of nucleotide sequence divergence was observed, ranging from 100% identity to
17
404
98.94% identity (5 nucleotide substitutions) between C. brevitarsis collected in Brisbane,
405
Australia and C. oxystoma (GenBank JN166962) collected in Israel.
406
Phylogenetic analysis of the 1,088 bp region of the gyrB gene also indicated that
407
the Australian Cardinium isolates cluster with group C Cardinium hertigii (Fig. S1b).
408
There was generally a higher level of sequence divergence in gyrB compared to 16S
409
rDNA but there was 100% identity observed between some Cardinium gyrB sequences.
410
Highest divergence (95.87% identity or 45 nucleotide substitutions) occurred between
411
Cardinium sequences from C. multimaculatus from Australia and C. imicola from Israel
412
(GenBank JN166963) and Kenya. In this analysis, the Cardinium sequences grouped
413
predominately to the geographical region from which the host Culicoides had been
414
collected, with the exception of C. williwilli from Brookfield in Queensland which were
415
most similar to Cardinium detected in C. arakawae from Japan (Fig. 2, Fig. S1b).
416
Translation of the gyrB gene sequences indicated that the majority (90%) of amino acid
417
substitutions in the gyrase B protein were synonymous.
418
A 1,563 bp amplicon assembled by concatenation of the 16S rDNA and gyrB
419
Cardinium genes (Fig. 2) generated a phylogeny consistent with the phylogenetic trees
420
constructed using the individual genes (Fig. S1a and S1b). Highest divergence of the
421
concatenated sequences (97.05% identity or 47 nucleotide substitutions) was between
422
C. multimaculatus from Australia and C. ohmorii from Japan, C. imicola from Israel and
423
C. imicola Kenya. The concatenated tree was assessed for the presence of
424
recombination using the program RDP v.4.39, but no putative recombination events
425
detected.
426
18
427
Wolbachia phylogenetic analysis. Phylogenetic analysis of Wolbachia (Fig. 3) based
428
on partial sequences of the 16S rDNA (390 bp amplicon) and the wsp gene (351 bp
429
amplicon), were generated through nested- and hemi-nested-PCR. Success of
430
amplifying sequences from low level Wolbachia infections was lower than for Cardinium
431
and only 16S or wsp amplicons were obtained for many samples. The 16S rDNA
432
sequences displayed a high level of homology to the only other Wolbachia sequence
433
reported previously from Culicoides which was from C. paraflavescens collected in
434
Japan (AB506794). The highest level of divergence from this sequence (98.72%
435
identity) was with Wolbachia from C. victoriae [241] collected in Australia. The wsp gene
436
sequences displayed generally lower levels of sequence identity with greatest
437
divergence (79.5% identity) observed in a group of C. narrabeenensis which were
438
collected in Brisbane, Australia. BLAST analysis of C. narrabeenensis samples showed
439
the closest sequence identity of 98% to Wolbachia isolated from Phengaris teleius
440
(JX470438) which is placed in Wolbachia supergroup B (45).
441 442
Occurrence of endosymbionts within the genus Culicoides and location.
443
Wolbachia and Cardinium detections were investigated across the Culicoides genus to
444
determine if there was a specific association with a particular subgenus. This was done
445
based on the morphological classification of Culicoides species, largely based on wing
446
classification (Table 5) (30). Wolbachia were detected sporadically across different
447
subgenera, with no obvious pattern identified (Table 5). Cardinium was seen to infect all
448
species except C. victoriae [172], with a low prevalence in other C. victoriae species.
449
This was further investigated by constructing a phylogenetic tree based on COI of the C.
450
victoriae group, with the absence of Cardinium not restricted to a particular lineage (Fig. 19
451
S2). As expected, Cardinium detections were more common across Culicoides species
452
than Wolbachia. Finally to determine if there was an association with the presence of
453
Wolbachia, Cardinium and geographical location, infection prevalence was plotted
454
against capture site (Fig. 1), with no obvious association apparent.
455 456
DISCUSSION
457 458
The genus Culicoides is one of the least studied of the major dipteran vector groups,
459
with limited information known of their endosymbionts. Recent studies have identified
460
Wolbachia and Cardinium in Culicoides species collected in Japan, Israel and the
461
United Kingdom. These studies used conventional PCR assays to reveal a relatively low
462
prevalence of both Wolbachia (1/34) and Cardinium (8/34) (13-15). In the present study,
463
20 species of Culicoides, predominately collected in south-eastern Australia, were
464
screened for the presence of these endosymbionts. Previously established screening
465
methodologies were utilized (13) to identify Cardinium in samples of C. victoriae [240],
466
C. victoriae [245], C. williwilli, C. imicola (Kenya) and C. brevitarsis; however these
467
methods failed to detect Wolbachia in individuals of any of the species tested. Bacteria
468
of the species Asaia were identified in C. marksi and C. brevitarsis samples by
469
conventional PCR screening utilizing alphaproteobacteria primers. Asaia species have
470
previously not been identified in Culicoides. Asaia has been detected in Anopheles
471
mosquitoes, colonizing the salivary glands and midgut of the insect (46). Unlike
472
Wolbachia and Cardinium, Asaia species can be cultured on medium and easily
473
colonize insects. Hence, these bacteria have been suggested as
474
paratransgenesis control agents of insect vectors (46, 47).
potential
20
475
Through the use of qPCR assays, previously undetected low level Cardinium and
476
Wolbachia infections were found in Culicoides species. Low level detections were due
477
to an improvement in detection sensitivity of approximately 100-fold gained through
478
utilizing qPCR assays, as compared to conventional PCR screening methodologies.
479
The qPCR screening identified a significant number of low level Wolbachia and
480
Cardinium infections in Culicoides with the improved sensitivity increasing detection
481
prevalence. Using conventional PCR, Nakamura et al. (2009) reported a Cardinium
482
infection prevalence of 16% in Culicoides but our analysis indicates that this is likely to
483
be an underestimate of the true prevalence. Following a similar pattern, Wolbachia has
484
previously only been detected in a single C. paraflavescens individual (13), whereas we
485
have detected Wolbachia in 7% of female and 5% of male Culicoides that were
486
screened.
487
This study also detected evidence of a range of low- and high-level infections in
488
Culicoides with no discernible pattern identified to explain this variability. High density
489
Cardinium infections detected by conventional PCR were found in C. brevitarsis, C.
490
victoriae [240, 245], C. williwilli and C. imicola Kenya with target copy numbers ranging
491
from ≈120,000 to 47,140,000 based on relative quantification. Low level infections only
492
detected by qPCR and confirmed by nested-PCR were found in a range from ≈125 to
493
60,000 target copies per Culicoides. Changes in endosymbiont density can be a result
494
of a range of factors, such as the bacterial strain (48, 49), host age (49, 50), sex (50)
495
and temperature (51, 52). Endosymbiont density based on Cq values were investigated
496
with Culicoides species that saw Cardinium infections in both sexes. No difference was
497
seen between sexes in C. bundyensis, C. austropalpalis and C. victoriae [240], whereas
498
C. parvimaculatus females had a higher density but this result is dependent on low 21
499
numbers (only 2 males and 8 females). Morag et al. (2012) provides evidence that
500
temperature could affect endosymbiont density, based on a lower prevalence of
501
Cardinium in Culicoides in arid regions than in Mediterranean regions. Low level
502
endosymbiont infections can be highly localized within the insect host, such as in some
503
Drosophila species (53), highlighting the importance of screening the whole insect
504
instead of using only abdomens (54). Although these infections occur at a low level,
505
they may still have a significant impact on their host insect, as has been shown for
506
Wolbachia in Drosophila paulistorum semi-species which can influence fecundity, sex
507
ratio and mate discrimination (23). Studies on the effect of Cardinium in C. imicola have
508
found no infection impact on Culicoides survival under optimal, starvation, heat and
509
antibiotic treatments, and no effect on wing size (55). However, detection in that study
510
involved conventional PCR screening and perhaps an effect on Cardinium was
511
overlooked because low level endosymbiont infections were not indentified or other
512
effects of Cardinium were not characterized.
513
Based on 16S rDNA and gyrB sequences, Cardinium hertigii was the only
514
Cardinium strain identified in this study. Specific to Culicoides, group C Cardinium has
515
previously been reported with
1
Novel detection of low level Cardinium and Wolbachia infections in Culicoides
2
Running title: Low level endosymbionts in Culicoides
3 4
Peter T Mee1,2, Andrew R Weeks1, Peter J Walker2, Ary A Hoffmann1, Jean-Bernard
5
Duchemin2#
6
Keywords: Culicoides; endosymbionts; biting midge; Cardinium; Wolbachia
7 8 9 10
1
Bio21 Institute and School of BioSciences, The University of Melbourne, 3052,
Parkville, Australia. 2
CSIRO Biosecurity, Australian Animal Health Laboratory, Geelong, 3219, Victoria,
Australia.
11 12 13 14 15 16
Corresponding author
17
Jean-Bernard Duchemin [email protected]
18 19 20 21 22 23 24 1
25
Abstract
26
Bacterial endosymbionts have been identified as potentially useful biological control
27
agents for a range of invertebrate vectors of disease. Previous studies of Culicoides
28
(Diptera: Ceratopogonidae) species using conventional PCR assays have provided
29
evidence of Wolbachia (1/33) and Cardinium (8/33) infections. Here, we screened 20
30
species of Culicoides, for Wolbachia and Cardinium, utilizing a combination of
31
conventional PCR and more sensitive qPCR assays. Low levels of Cardinium DNA were
32
detected in females of all but one of the Culicoides species screened and low levels of
33
Wolbachia were detected in females of 9 of the 20 Culicoides species. Sequence
34
analysis based on partial 16S rDNA and gryB identified Candidatus Cardinium hertigii
35
from group C, which has previously been identified in Culicoides from Japan, Israel and
36
the United Kingdom. Wolbachia strains detected in this study showed 98-99% sequence
37
identity to Wolbachia previously detected from Culicoides based on 16S rDNA, whereas
38
a strain with a novel wsp sequence was identified in C. narrabeenensis. Cardinium
39
isolates grouped to geographical regions independent of host Culicoides species,
40
suggesting possible geographical barriers to Cardinium movement. Screening also
41
identified the bacteria Asaia in Culicoides. These findings point to a diversity of low level
42
endosymbiont infections in Culicoides, providing candidates for further characterization
43
and highlighting the widespread occurrence of these endosymbionts in this insect group.
44 45 46 47 48 2
49
INTRODUCTION
50
Culicoides are small haematophagous insects (Diptera: Ceratopogonidae), some of
51
which are important vectors of viral and parasitic diseases of veterinary and medical
52
importance (1). More than 50 different viruses have been isolated from Culicoides
53
species, including bluetongue virus (BTV), Schmallenberg virus (SBV) and African
54
horse sickness virus (AHSV) which cause significant impacts on livestock production
55
through stock losses and trade restrictions (1). Capable of wind-borne displacement for
56
several hundred kilometers, Culicoides have a capacity for rapid long distance
57
transmission of disease and have recently been responsible for the establishment of
58
enzootic BTV and SBV infections over vast new geographic areas (1, 2). Current control
59
methods for Culicoides include breeding site removal, baiting of livestock and midge
60
resting sites; however these techniques are costly, labor intensive and have varying
61
levels of success and permanence of control (3). Although vaccines are available for
62
some Culicoides-transmitted viruses, such as BTV, the practicality of vaccination is
63
limited by the large number of BTV serotypes and the potential for genome segment
64
reassortment between live vaccines and naturally circulating virus strains (4).
65
Inactivated vaccines are expensive and less potent but have been used effectively in
66
Europe for control of BTV (5). Inactivated vaccines are not currently available for AHSV.
67
Insect vector control by use of endosymbiotic organisms has gained increasing
68
attention in recent years (6). Bacterial endosymbionts present in arthropod species are
69
capable of influencing host characteristics such as longevity and vector competence, as
70
well as being involved in nutrient provisioning (7-9). The endosymbiont Wolbachia
71
pipientis (Alphaproteobacteria) has attracted notable attention for its applicability to
72
endosymbiont-based control of the dengue virus vector, Aedes aegypti. Wolbachia has 3
73
been shown to successfully invade and be maintained in natural A. aegypti populations
74
and to block virus transmission (8, 10-12).
75
Several previous studies have reported evidence of bacterial endosymbiont
76
infection in Culicoides species. Screening studies conducted using conventional PCR
77
assays detected Wolbachia DNA in a single Culicoides paraflavescens individual in
78
Japan
79
endosymbiont which also has a range of influences on its host insect, has been
80
detected in four Culicoides species in Japan, two Culicoides species in Israel and two in
81
the United Kingdom (13-15). Endosymbiotic diversity in Australian Culicoides species
82
has not been investigated previously, nor has a comparative analysis of Cardinium
83
divergence in different Culicoides species from diverse geographical locations been
84
reported.
(13).
Candidatus
Cardinium
hertigii
(Bacteroidetes),
another
bacterial
85
Although conventional PCR has previously been used successfully to screen
86
arthropods for endosymbionts (16-18), recent studies have demonstrated that this
87
method can fail to detect low level infections. More sensitive screening techniques such
88
as long-(19), nested-(20) or quantitative-PCR (21) are therefore required. Low level
89
endosymbiont infections have been identified in a range of insects from tsetse flies (22),
90
Drosophila (23), Cherry fruit fly (24) and planthoppers (25). Previous studies have
91
suggested that at low levels of infection endosymbionts are capable of influencing the
92
host. For example, low levels of Wolbachia in Drosophila paulistorum semispecies have
93
been shown to influence fecundity, sex ratios and mate discrimination (23). However
94
other endosymbiont effects including viral blockage and fitness effects may depend on
95
bacterial density (26).
4
96
In this study, a range of Culicoides species, collected predominately from south-
97
eastern Australia, were screened for evidence of Cardinium and Wolbachia infection.
98
Global movement of Cardinium in Culicoides species was also investigated based on
99
sequence divergence in multiple loci. Novel Cardinium and Wolbachia infections were
100
identified in a range of Culicoides species, a high proportion of which were low-level
101
infections. Nucleotide sequence analysis revealed that Cardinium detected in these
102
samples was genetically similar to those previously discovered in Japan, Israel and the
103
United Kingdom, suggesting a global presence of a single Cardinium strain throughout a
104
wide geographical range and in a range of Culicoides species.
105 106
MATERIALS AND METHODS
107 108
Insect collection. Culicoides were collected using either Centre for Disease Control
109
(CDC) mini light traps (BioQuip Products, Rancho Dominguez, CA) or yellow sticky
110
traps (YST). Mini light traps contained either green or ultraviolet LEDs and were
111
powered by a 6 V motorbike battery (27, 28). Traps were positioned approximately 2 m
112
above the ground at dusk and collected after dawn, consistent with previously trapping
113
methodologies (29). A downdraught fan in the traps directed insects into a beaker
114
containing approximately 200 ml of 80% ethanol. Contents of the beakers were
115
collected daily. Before storing at -20 °C, insects were sorted, identified and stored
116
individually in fresh 80% ethanol. By washing insects in ethanol, the risk of
117
contamination was reduced. Yellow sticky traps (YST) (Trappit) were elevated
118
approximately 10 cm above the ground by a plastic stick and illuminated by a solar
119
garden light. Insects were individually cut from the YST and placed in a tube containing 5
120
enough solvent (De-Solv-it®, orange oil) (Orange-Sol, Chandler, AZ) to cover the
121
sample. Tubes were incubated at 60 oC for 5 min with intermittent inversion until the
122
insect floated off the YST. Insects were removed and washed twice with 80% ethanol
123
before storing in fresh 80% ethanol at -20 oC (I. Valenzuela pers. comm.). Collections
124
were made from 305 traps (81 CDC & 224 YST) over a period of 33 trapping nights
125
throughout south-eastern Australia from January to April in 2013 and 2014. Samples of
126
C. imicola collected using CDC light traps were also obtained from Kenya and
127
Madagascar, through the International Livestock Research Institute, Kenya. Culicoides
128
imicola were identified morphologically based on wing patterning, and through sequence
129
analysis of the cytochrome oxidase subunit 1 (COI) region.
130 131
Culicoides identification. Morphological identification was performed with the aid of
132
the pictorial atlas of Australasian Culicoides wings (Diptera: Ceratopogonidae) (30). The
133
C. victoriae species group was separated into a series of species denoted by wing
134
identifier number in square brackets, such as C. victoriae [241], based on the wing
135
pictures from Dyce et al. (2007). One C. victoriae species not present in pictorial atlas
136
was collected in Geelong, Victoria and is denoted as C. victoriae [172] (BOLD sequence
137
ID CUVIC12-15). Genetic confirmation of Culicoides species was based on amplification
138
and sequencing of the COI, which is commonly used for species determination of
139
Culicoides and capable of differentiating most Culicoides species (31, 32).
140 141
DNA extractions. Culicoides were removed from ethanol and air dried before extraction
142
(33). The mouse tail protocol of the MagMaxTM-96 DNA Multi-Sample Kit (Applied
143
Biosystems, Austin, TX) on a KingFisherTM Flex (Thermo Scientific, Waltham, MA) was 6
144
used with the following amendments. Individual Culicoides were incubated in 100 µl of
145
PK buffer and 10 mg/ml of Proteinase K. Samples were homogenized with a
146
FastPrep®-24 Instrument (MP Biomedicals, Santa Ana, CA) at 6.0 m/s for 20 s with
147
approximately 10 x 1.0 mm zirconia/silica beads (BioSpec Products, Bartlesville, OK)
148
before overnight incubation at 56 oC. Elution was altered to 40 µl of both elution buffer 1
149
and 2. To assess possible contamination during extraction every eleventh well in the
150
MagMaxTM 96-well plate was left without a Culicoides sample, but was still included in
151
all downstream screening steps.
152 153
Conventional PCR screening. Primers (GeneWorks, SA, Australia) utilized included
154
an initial housekeeper targeting the third loop of the 28S ribosomal DNA (rDNA) (D3a,
155
D3ba),
156
Alphaproteobacteria 16S rDNA (Alf28F, Alf684R) (34, 35), Wolbachia 16S rDNA (Wol-F,
157
Wol-R) (36) and cytochrome oxidase subunit 1 (COI) (BC1culicFm, JerR2m) (32), for
158
species confirmation. Primer sequences can be found in Table 1. 25 µL PCR reactions
159
were performed using 1 unit Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad,
160
CA), following manufacturer’s protocol. PCR conditions were as follows: 1 cycle 95 oC
161
for 2 mins; 35 cycles of 95 oC for 30 sec, anneal temperature as in Table 1 for 30 sec,
162
72 oC for 30 sec; and final extension at 72 oC for 5 min. Two negative controls of ultra
163
pure water were included for all PCR screens to ensure no contamination occurred. A
164
single positive control was included for each PCR, DNA from C. victoriae [240] was
165
used for 28S and COI PCR, and DNA from Drosophila simulans was used for
166
Wolbachia 16S and Alphaproteobacteria 16S PCR. At the commencement of screening
167
no positive control was available for Cardinium; in later steps C. victoriae [240] samples
followed
by
Cardinium
16S
rDNA
(CAR-SP-F,
CAR-SP-R)
(13),
7
168
shown to be Cardinium-positive were used as a control. Amplified products were
169
separated on a 1% agarose gel containing 0.1% SYBR® Safe DNA Gel Stain
170
(Invitrogen, USA) and visualized with a G:BOX Syngene blue light visualization
171
instrument.
172 173
Multi-allele typing. Samples which tested positive for Cardinium by conventional PCR
174
screening were typed for multiple alleles using 16S rDNA and DNA gyrase subunit B
175
(gyrB). Four primers (16SA1F, 16SA1R, 16SA2F & 16SA2R) (Table 2) were designed
176
to amplify two overlapping regions of Cardinium 16S rDNA, based on the Clustal W
177
alignment of sequences from Culicoides (AB506776-AB506779, JN166961, JN166962)
178
available in the GenBank database. Primers were designed using Geneious v7.0.5, and
179
covered 1,404 bp of the 16S rDNA gene, with 243 bp overlap between the amplicons
180
(37). Four primers (gyrBA1F, gyrBA1R, gyrBA2F & gyrBA2R) targeting a 1,399 bp
181
region of gyrB gene with 168 bp overlap between amplicons (Table 2) were designed
182
using the same process as above, with sequences available in GenBank (AB506791,
183
AB506792, JN166963, JN166964). PCR reactions were performed using the conditions
184
as previously stated, an annealing temperature of 50 °C was used for Cardinium 16S
185
rDNA and gyrB primers.
186
Low level Wolbachia detections were amplified using a combination of hemi-
187
nested (16S) and nested (wsp) PCRs. Hemi-nested primers for 16S used two sets of
188
previously published primers from Wol-F and Wol-R (36) and Rao-F and Rao-R (38).
189
First round amplification was performed using the Wol-F and Wol-R as previously
190
outlined, generating an 897 bp amplicon. Second round amplification yielded two
191
amplicons, amplicon 1 = 464 bp (Wol-F and Rao-R) and amplicon 2 = 495 bp (Rao-F 8
192
and Wol-R); both rounds of amplification had an annealing temperature of 52 oC. A
193
nested-PCR developed by Hughes et al. (2012) (Table 1) was used to amplify a 423 bp
194
region of the wsp. The nested protocol was altered to a two-step nested PCR instead of
195
the previously outlined single tube nested-PCR (39). Two negative controls both
196
involving ultra-pure water as template were run for first round amplification, with the
197
product used as template for second round amplification.
198 199
Quantitative PCR screening. Quantitative PCR (qPCR) was used to increase the level
200
of sensitivity of detection of Wolbachia and Cardinium. Samples screened with the
201
conventional assays were rescreened with the qPCR assays. A Wolbachia qPCR was
202
obtained based on the primer and probe design of Rao et al. (2006), which used this
203
assay to detect Wolbachia in field collected mosquitoes. The probe reporter dye at the
204
5’ end was changed to VIC (4, 7, 2’-trichloro-7’-phenyl-6-carboxyfluorescein) as
205
opposed to FAM (6-carboxyfluorescein) but retained the same TAMRA (6-
206
carboxytetramethylrhodamine) quencher dye at the 3’ end. Wolbachia primers and the
207
probe reaction setup was modified to a 20 µl reaction using 10 µl of TaqMan® Universal
208
PCR Master Mix (Applied Biosystems, Foster City, CA), 1 µl of both primers at 0.3 µM
209
forward, 0.9 µM reverse, 1 µl of 0.05 µM probe, 2 µl of template DNA and 5 µl of ultra-
210
pure water.
211
To develop a Cardinium qPCR, published Cardinium sequences isolated from
212
Culicoides (AB506776-AB506779, JN166961 and JN166962) were aligned using
213
Geneious v7.0.5 and a consensus sequence obtained. Primer Express® software
214
v3.0.1. (Applied Biosystems) was used to design a TaqMan probe and primer pair to
215
amplify a 111 bp region (Table 3) to specifically target Cardinium group C. Cardinium 9
216
primers and probes were calibrated to a final reaction volume of 20 µl consisting of 10 µl
217
of TaqMan® Universal PCR Master Mix, 2 µl of 0.3 µM forward and reverse primer, 2 µl
218
of 0.05 µM probe, 2 µl of template DNA and ultra-pure water. Optimal primer and probe
219
concentrations for both assays were determined as in the protocol for the TaqMan®
220
Universal PCR Master Mix. Triplicate reactions were performed on a QuantStudioTM 6
221
real-time PCR machine (Life Technologies, Carlsbad, CA). Cycling conditions included
222
an initial incubation at 50 oC for 2 min to activate uracil-N-glycosylase, followed by
223
denaturation 10 min at 95 oC then 45 cycles of 95 oC for 15 s and a combined annealing
224
and primer extension phase at 60 oC for 1 min. Screening was conducted in a 96 well
225
plate format with 6 wells each plate being left for use as negative controls.
226
Cloned standards of Cardinium and Wolbachia were generated and used as
227
positive controls, as follows. Conventional PCR was conducted using qPCR primers to
228
amplify known Wolbachia positive material from Drosophila simulans and Cardinium
229
positive material from Culicoides victoriae [240]. Amplicons were visualized on a 1%
230
agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA).
231
Purified DNA was ligated into pGEM®-T Easy Vector (Promega, Madison, WI)
232
according to the manufacturer’s protocol, then electroporated into competent E. coli
233
DH5α cells (New England Biolabs, Beverly, MA). Plasmid DNA was purified from
234
positive clones using QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA) and sequenced
235
using SP6 and T7 sequencing primers. Plasmid DNA clones were serially diluted before
236
being used as positive controls. Wolbachia and Cardinium plasmids was also used to
237
generate a standard regression curve based on 6 serial dilutions of the plasmid in each
238
qPCR run, allowing relative quantification of infection. Plasmid copy numbers were
10
239
calculated
as
per
established
240
(http://www6.appliedbiosystems.com/support/tutorials) (Applied Biosystems).
protocols
241 242
Sensitivity comparison of conventional and qPCR. Detection limits of conventional
243
and quantitative PCR were assessed by serially diluting Wolbachia positive template
244
DNA from Drosophila simulans and Cardinium positive template DNA from Culicoides
245
victoriae [240]. Ultra-pure water was used to make 10-fold serial dilutions of each DNA
246
template from neat to 10-9. Duplicate 2 µl samples of each dilution were tested using the
247
Cardinium conventional (CAR-SP-F, CAR-SP-R) (13) and quantitative assays (Table 3),
248
as well as the Wolbachia conventional (Wol-F, Wol-R) (36) and quantitative assays
249
(Table 3).
250 251
Sequencing. Nucleotide sequences of Cardinium, Wolbachia and Alphaproteobacteria
252
positive amplicons were determined for species confirmation. DNA was purified from
253
positive amplicons with a QIAquick Gel Extraction Kit (Qiagen, Santa Clara, CA)
254
according to the manufacturer’s protocol. Fluorometric quantification of purified DNA
255
was performed using a dsDNA HS Assay on a Qubit® (Invitrogen, Carlsbad, CA).
256
Sequencing was performed using the BigDye® Terminator v3.1 kit (Applied Biosystems,
257
Foster City, CA) according to the manufacturer’s protocol and sequence was generated
258
via capillary Sanger sequencing on a 3500 Genetic Analyzer (Applied Biosystems).
259
A series of measures was implemented as an added precaution to reduce the
260
risk of contamination and to ensure no false-positive detections. Clean-room working
261
conditions were utilized with insect identification, nucleotide extraction, master mix set
11
262
up, amplification and post-amplification handling all being performed in separate labs,
263
with a non-amplified to amplified workflow being followed.
264
To compare the incidence of infections across sexes, contingency analyses were
265
carried out for species where the infection was detected in at least one sex. Probabilities
266
for detecting associations between infections and sex were based on exact tests.
267 268
Phylogenetic analysis. Phylogenetic relationships of Cardinium were analyzed using
269
nucleotide sequences obtained from 16S rDNA and gyrB genes. Additional group A
270
(isolated from Ixodes scapularis (AB001518, AB506790), Euides speciosa (AB506775,
271
AB506788), Tetranychus pueraricola (AB241135, AB506784), Oligonychus ilicis
272
(AB241130, AB506783)) and group B (isolated from Paenicardinium endonii
273
(DQ314214, DQ314215)) Cardinium sequences obtained from GenBank were included
274
to provide phylogenetic resolution. Cardinium 16s rDNA and gyrB sequences were
275
concatenated head-to-tail forming a super-gene alignment. Concatenating two genes
276
together increases the number of evolutionary changes on individual branches which in
277
turn can generate a more precise phylogeny, as opposed to when the two genes are
278
analyzed separately (40). The concatenated alignment was screened for the presence
279
of recombination using the RDP, GENECONV, Bootscan, MaxChi, Chimaera and
280
SiScan methods implemented in Recombination Detection Program v4.39 (RDP4), with
281
default parameters (41). A Wolbachia phylogeny was determined using 16S rDNA and
282
Wolbachia Surface Protein (wsp). The wsp gene was included in phylogenetic analysis
283
as it has been shown to have a much greater divergence rate than 16S rDNA (18, 36).
284
Wolbachia sequences from supergroup A (Drosophila sechellia), B (Culex pipiens) and
285
C (Dirofilaria immitis) were included to see where Culicoides Wolbachia would be 12
286
placed within the wider Wolbachia phylogeny, supergroup C being used to root the
287
trees. Host Culicoides phylogenetic analysis was based on mitochondrial cytochrome
288
oxidase subunit I (COI).
289
All sequences were aligned with the Clustal W algorithm in Geneious v7.0.5
290
(Biomatters Ltd) (37). Aligned nucleotide sequences were analyzed using jModelTest2
291
v2.1.3, with the topology search taking the best of Nearest Neighbor Interchange (NNI)
292
and Subtree Pruning and Regrafting (SPR) (42). Evolutionary models implemented to
293
construct phylogenetic trees were selected based on the Akaike Information Criterion
294
output from JModelTest2 (42) (number of substitution schemes 11, base tree for
295
likelihood calculations Maximum-Likelihood (ML) optimized, base tree search best). The
296
Hasegawa-Kishino-Yano (HKY) model was selected for Cardinium 16S rDNA,
297
Wolbachia wsp and the concatenation of Cardinium 16S rDNA and gyrB. The General
298
Time-Reversible (GTR) model was selected for the Cardinium gyrB, Wolbachia 16S
299
rDNA and Culicoides COI phylogeny. Phylogenetic ML trees were constructed using the
300
PhyML plugin in Geneious v7.0.5 with 1,000 bootstrap replicates; the proportion of
301
invariable sites and gamma distribution were both estimated (43).
302 303
Nucleotide sequence accession numbers. Sequences generated were deposited in
304
GenBank. Cardinium accession numbers for 16S rRNA are as follows KR026906-
305
KR0269018, KR0269020-KR026923, and gyrB gene are KR026924-KR026935.
306
Wolbachia accession numbers for 16S rRNA are KR026936-KR026941 and wsp are
307
KR026942-KR026951. Sequences for the Culicoides victoriae group were deposited in
308
Barcode of Life Data Systems (BOLD) database under the sample ID’s CUVIC10-15 to
309
CUVIC18-15. 13
310 311
Mapping of Cardinium and Wolbachia in Culicoides. Culicoides which tested
312
positive for Wolbachia or Cardinium based on screening with the quantitative assays,
313
were plotted against capture location. Shapefiles were obtained from the Australian
314
Bureau of Statistics, Australian Standard Geographical Classification (ASGC) Digital
315
Boundaries, Australia July 2011. The distribution map was created in quantum GIS
316
(QGIS) Wien v2.8.1 (44).
317 318
RESULTS
319 320
Endosymbiont assays. Increased sensitivity for the Wolbachia and Cardinium
321
quantitative PCRs was obtained in comparison to the conventional assays. Serial
322
dilution of Wolbachia and Cardinium positive samples saw a 100-fold lower detection
323
limit by the qPCR assays. Detections were confirmed by sequencing, however the
324
Wolbachia (62 bp of 16S) and Cardinium (111 bp of 16S) qPCR amplicons were too
325
small for species discrimination. Developed nested-PCRs for Wolbachia (16S and wsp)
326
and Cardinium (16S and gyrB) were used to amplifying enough material of the level low
327
detections to allow confirmation by sequencing.
328
Twenty Culicoides species were identified by morphological and genetic typing of
329
insects collected in Victoria, Tasmania and Queensland from January to April 2013 and
330
2014 (Fig. 1). These samples, together with samples of C. imicola from Kenya and
331
Madagascar, were analyzed for evidence of Cardinium and Wolbachia infection through
332
the conventional and quantitative PCR assays.
333 14
334
Cardinium screening. The conventional PCR assay identified evidence of Cardinium
335
infection in females of three species, C. victoriae [240] and C. brevitarsis from Australia,
336
and C. imicola from Kenya (Table 4). Rescreening the samples using the qPCR assay
337
indicated a substantially higher prevalence of Cardinium infection, with positive samples
338
detected in all Culicoides species except for C. victoriae [172] (Table 4). The percentage
339
of positives detected in females increased from 4% (14/360, CI: 2.23 - 6.28%)
340
(conventional PCR) to 26% (92/360, CI: 21.25 - 30.25%) (qPCR), and detections in
341
males increased from 0% (0/161, CI: 0 - 1.84%) (conventional PCR) to 14% (22/161, CI:
342
8.98-19.64%) (qPCR). A lower number of male Culicoides were screened due to their
343
lower representation in collections. However, in those Culicoides species in which
344
Cardinium was detected in individuals of both sexes, there was no significant difference
345
between prevalence in males and females for C. brevitarsis (contingency analysis, P =
346
0.63, df = 1), C. bundyensis (contingency analysis, P = 0.33, df = 1), and C. victoriae
347
[245] (contingency analysis, P = 0.24, df = 1), although differences were marginally non-
348
significant in the case of C. parvimaculatus (contingency analysis, P = 0.065, df = 1) and
349
C. austropalpalis (contingency analysis, P = 0.067, df = 1) due to a higher prevalence in
350
female Culicoides . For C. marksi no infection was identified in males but it was present
351
in some females (contingency analysis, P = 0.035).
352
Detections of Cardinium by conventional PCR were seen at the lowest Cq values of
353
26.58 in C. williwilli (equating to 23,000,000 copies) and the highest Cq value of 34.06
354
(equating to 24,000 copies) in C. victoriae [240] (Table S1). However, the Cardinium
355
qPCR assay provided detections at the lowest Cq values of 42.68 in C. parvimaculatus
356
which equates to approximately 290 copies of the Cardinium target region. In Culicoides
357
species which had detections of Cardinium in both sexes, there was no significant 15
358
difference (based on t-tests) in Cq values and hence Cardinium copy number between
359
sexes in C. bundyensis (P = 0.22, df = 10) and C. austropalpalis (P = 0.29, df = 8),
360
although there was a difference for C. parvimaculatus (P = 0.018, df = 8).
361 362
Wolbachia screening. No evidence of Wolbachia infection was detected in any of the
363
Culicoides samples tested using the conventional PCR. In contrast, evidence of low-
364
level Wolbachia infections was detected in ten species of Culicoides when individual
365
samples were screened using the qPCR assay (Table 4). Across all species tested, the
366
percentages of detected positive females (7%, 23/353, CI = 4.27 – 9.46) and males
367
(5%, 8/160, CI = 2.34 – 9.27) were similar (contingency test, P = 0.55, df = 1).
368
Culicoides bundyensis was the only species in which evidence of Wolbachia infection
369
was detected in both sexes, with a similar proportion of positive males and females
370
(contingency test, P = 0.54, df = 1).
371
Detections of Wolbachia by qPCR were seen to range from the lowest Cq value
372
of 30.53 in C. antennalis (equating to 11,600 copies) and the highest Cq value of 41.30
373
(equating to 22.4 copies) in C. brevitarsis (Table S1). Detections of Wolbachia were
374
rarely seen in both sexes of the same Culicoides species, hence no comparison of Cq
375
value and sex were performed.
376
There was evidence of low-level dual infections with Wolbachia and Cardinium in
377
a number of Culicoides species. These included five individuals of C. antennalis, four
378
individuals of C. brevitarsis and C. parvimaculatus, three individuals of C. bundyensis
379
and C. victoriae [245], and single individuals of C. imicola (Madagascar), C. marksi, C.
380
austropalpalis and C. marmoratus (Table 5). The dual infections in single individuals
16
381
occurred at the expected frequency on the basis of a random association between the
382
infections.
383 384
Alphaproteobacteria screening. Screening by conventional PCR was also performed
385
using the broad range Alphaproteobacteria primer set. The assay provided evidence of
386
infection in individuals representing four Culicoides species: C. austropalpalis in which
387
30% (13/43, CI = 17.96 – 45.09%) females and 11% (3/28, CI = 2.79 – 26.45%) males
388
were positive; C. marksi in which 7% (2/29, CI = 1.17 – 20.97%) females and 5% (1/19,
389
CI = 0.26 – 23.33%) of males were positive; C. brevitarsis in which 22% (2/9, CI = 3.90
390
– 56.21%) females and 50% (1/2, CI = 2.5 – 97.5%) males were positive; and C.
391
bundyensis in which 18% (5/27, CI = 7.11 – 36.38%) females were positive. The
392
amplicons generated in the assay were sequenced. BLAST analysis indicated that
393
amplicons obtained from C. marksi and C. brevitarsis shared a high percentage of
394
nucleotide sequence identity (99%) with the 16S ribosomal DNA gene of an Asaia sp.
395
bacterium isolated from Aedes albopictus mosquitoes (Genbank JX445140.1).
396 397
Cardinium phylogenetic analysis. Partial 16S ribosomal DNA and DNA gyrase
398
subunit B gene sequences were used to construct the Cardinium phylogeny. Based on
399
the 505 bp 16S rDNA amplicon, all Australian Cardinium isolates grouped with the
400
previously described sequences of Culicoides group C Cardinium hertigii from Israel (C.
401
oxystoma and C. imicola), Japan (C. arakawae, C. lungchiensis, C. peregrinus and C.
402
ohmorii) and the United Kingdom (C. punctatus and C. pulicaris) (Fig. S1a). A low level
403
of nucleotide sequence divergence was observed, ranging from 100% identity to
17
404
98.94% identity (5 nucleotide substitutions) between C. brevitarsis collected in Brisbane,
405
Australia and C. oxystoma (GenBank JN166962) collected in Israel.
406
Phylogenetic analysis of the 1,088 bp region of the gyrB gene also indicated that
407
the Australian Cardinium isolates cluster with group C Cardinium hertigii (Fig. S1b).
408
There was generally a higher level of sequence divergence in gyrB compared to 16S
409
rDNA but there was 100% identity observed between some Cardinium gyrB sequences.
410
Highest divergence (95.87% identity or 45 nucleotide substitutions) occurred between
411
Cardinium sequences from C. multimaculatus from Australia and C. imicola from Israel
412
(GenBank JN166963) and Kenya. In this analysis, the Cardinium sequences grouped
413
predominately to the geographical region from which the host Culicoides had been
414
collected, with the exception of C. williwilli from Brookfield in Queensland which were
415
most similar to Cardinium detected in C. arakawae from Japan (Fig. 2, Fig. S1b).
416
Translation of the gyrB gene sequences indicated that the majority (90%) of amino acid
417
substitutions in the gyrase B protein were synonymous.
418
A 1,563 bp amplicon assembled by concatenation of the 16S rDNA and gyrB
419
Cardinium genes (Fig. 2) generated a phylogeny consistent with the phylogenetic trees
420
constructed using the individual genes (Fig. S1a and S1b). Highest divergence of the
421
concatenated sequences (97.05% identity or 47 nucleotide substitutions) was between
422
C. multimaculatus from Australia and C. ohmorii from Japan, C. imicola from Israel and
423
C. imicola Kenya. The concatenated tree was assessed for the presence of
424
recombination using the program RDP v.4.39, but no putative recombination events
425
detected.
426
18
427
Wolbachia phylogenetic analysis. Phylogenetic analysis of Wolbachia (Fig. 3) based
428
on partial sequences of the 16S rDNA (390 bp amplicon) and the wsp gene (351 bp
429
amplicon), were generated through nested- and hemi-nested-PCR. Success of
430
amplifying sequences from low level Wolbachia infections was lower than for Cardinium
431
and only 16S or wsp amplicons were obtained for many samples. The 16S rDNA
432
sequences displayed a high level of homology to the only other Wolbachia sequence
433
reported previously from Culicoides which was from C. paraflavescens collected in
434
Japan (AB506794). The highest level of divergence from this sequence (98.72%
435
identity) was with Wolbachia from C. victoriae [241] collected in Australia. The wsp gene
436
sequences displayed generally lower levels of sequence identity with greatest
437
divergence (79.5% identity) observed in a group of C. narrabeenensis which were
438
collected in Brisbane, Australia. BLAST analysis of C. narrabeenensis samples showed
439
the closest sequence identity of 98% to Wolbachia isolated from Phengaris teleius
440
(JX470438) which is placed in Wolbachia supergroup B (45).
441 442
Occurrence of endosymbionts within the genus Culicoides and location.
443
Wolbachia and Cardinium detections were investigated across the Culicoides genus to
444
determine if there was a specific association with a particular subgenus. This was done
445
based on the morphological classification of Culicoides species, largely based on wing
446
classification (Table 5) (30). Wolbachia were detected sporadically across different
447
subgenera, with no obvious pattern identified (Table 5). Cardinium was seen to infect all
448
species except C. victoriae [172], with a low prevalence in other C. victoriae species.
449
This was further investigated by constructing a phylogenetic tree based on COI of the C.
450
victoriae group, with the absence of Cardinium not restricted to a particular lineage (Fig. 19
451
S2). As expected, Cardinium detections were more common across Culicoides species
452
than Wolbachia. Finally to determine if there was an association with the presence of
453
Wolbachia, Cardinium and geographical location, infection prevalence was plotted
454
against capture site (Fig. 1), with no obvious association apparent.
455 456
DISCUSSION
457 458
The genus Culicoides is one of the least studied of the major dipteran vector groups,
459
with limited information known of their endosymbionts. Recent studies have identified
460
Wolbachia and Cardinium in Culicoides species collected in Japan, Israel and the
461
United Kingdom. These studies used conventional PCR assays to reveal a relatively low
462
prevalence of both Wolbachia (1/34) and Cardinium (8/34) (13-15). In the present study,
463
20 species of Culicoides, predominately collected in south-eastern Australia, were
464
screened for the presence of these endosymbionts. Previously established screening
465
methodologies were utilized (13) to identify Cardinium in samples of C. victoriae [240],
466
C. victoriae [245], C. williwilli, C. imicola (Kenya) and C. brevitarsis; however these
467
methods failed to detect Wolbachia in individuals of any of the species tested. Bacteria
468
of the species Asaia were identified in C. marksi and C. brevitarsis samples by
469
conventional PCR screening utilizing alphaproteobacteria primers. Asaia species have
470
previously not been identified in Culicoides. Asaia has been detected in Anopheles
471
mosquitoes, colonizing the salivary glands and midgut of the insect (46). Unlike
472
Wolbachia and Cardinium, Asaia species can be cultured on medium and easily
473
colonize insects. Hence, these bacteria have been suggested as
474
paratransgenesis control agents of insect vectors (46, 47).
potential
20
475
Through the use of qPCR assays, previously undetected low level Cardinium and
476
Wolbachia infections were found in Culicoides species. Low level detections were due
477
to an improvement in detection sensitivity of approximately 100-fold gained through
478
utilizing qPCR assays, as compared to conventional PCR screening methodologies.
479
The qPCR screening identified a significant number of low level Wolbachia and
480
Cardinium infections in Culicoides with the improved sensitivity increasing detection
481
prevalence. Using conventional PCR, Nakamura et al. (2009) reported a Cardinium
482
infection prevalence of 16% in Culicoides but our analysis indicates that this is likely to
483
be an underestimate of the true prevalence. Following a similar pattern, Wolbachia has
484
previously only been detected in a single C. paraflavescens individual (13), whereas we
485
have detected Wolbachia in 7% of female and 5% of male Culicoides that were
486
screened.
487
This study also detected evidence of a range of low- and high-level infections in
488
Culicoides with no discernible pattern identified to explain this variability. High density
489
Cardinium infections detected by conventional PCR were found in C. brevitarsis, C.
490
victoriae [240, 245], C. williwilli and C. imicola Kenya with target copy numbers ranging
491
from ≈120,000 to 47,140,000 based on relative quantification. Low level infections only
492
detected by qPCR and confirmed by nested-PCR were found in a range from ≈125 to
493
60,000 target copies per Culicoides. Changes in endosymbiont density can be a result
494
of a range of factors, such as the bacterial strain (48, 49), host age (49, 50), sex (50)
495
and temperature (51, 52). Endosymbiont density based on Cq values were investigated
496
with Culicoides species that saw Cardinium infections in both sexes. No difference was
497
seen between sexes in C. bundyensis, C. austropalpalis and C. victoriae [240], whereas
498
C. parvimaculatus females had a higher density but this result is dependent on low 21
499
numbers (only 2 males and 8 females). Morag et al. (2012) provides evidence that
500
temperature could affect endosymbiont density, based on a lower prevalence of
501
Cardinium in Culicoides in arid regions than in Mediterranean regions. Low level
502
endosymbiont infections can be highly localized within the insect host, such as in some
503
Drosophila species (53), highlighting the importance of screening the whole insect
504
instead of using only abdomens (54). Although these infections occur at a low level,
505
they may still have a significant impact on their host insect, as has been shown for
506
Wolbachia in Drosophila paulistorum semi-species which can influence fecundity, sex
507
ratio and mate discrimination (23). Studies on the effect of Cardinium in C. imicola have
508
found no infection impact on Culicoides survival under optimal, starvation, heat and
509
antibiotic treatments, and no effect on wing size (55). However, detection in that study
510
involved conventional PCR screening and perhaps an effect on Cardinium was
511
overlooked because low level endosymbiont infections were not indentified or other
512
effects of Cardinium were not characterized.
513
Based on 16S rDNA and gyrB sequences, Cardinium hertigii was the only
514
Cardinium strain identified in this study. Specific to Culicoides, group C Cardinium has
515
previously been reported with
