Supporting information
DNA sequencing, primer design and PCR analysis of faecal samples
Total DNA was extracted from tissue samples from cricket legs using QIAGEN DNeasy
Tissue Kits (QIAGEN Inc., Chatsworth, California, USA) following the manufacturer’s animal
tissue protocol. To obtain sequences to facilitate cricket-specific primer design, polymerase
chain reaction (PCR) was performed to amplify cytochrome c oxidase subunit I (COI) from
camel and cave crickets [C. pallidipes, C. gracilipes (Haldeman), C. guttulosus F. Walker and
Euhadenoecus puteanus (Scudder)] using the primers LCO-1490 (Folmer et al., 1994) and HCO700ME (Breton et al. 2006). PCR reactions (50 μL) consisted of 1X Takara buffer (Takara Bio
Inc., Shiga, Japan), 0.2 mM of each dNTP, 0.2 mM of each primer, 1.25 U Takara Ex Taq™ and
template DNA (2 μL of total DNA). PCR reactions were carried out in Bio-Rad PTC-200 and
C1000 thermal cyclers (Bio-Rad Laboratories, Hercules, California, USA). The PCR cycling
protocols were 94 °C for 1 min followed by 50 cycles of 94 °C for 50 s, 40 °C for 45 s, 72 °C for
45 s and a final extension of 72 °C for 5 min. Reaction success was determined by
electrophoresis of 10 μL of PCR product in 1.5% SeaKem agarose (Lonza, Rockland, Maine,
USA) stained with ethidium bromide (0.1 mg/L). PCR reactions that yielded significant product
were purified with QIAGEN MinElute PCR purification kit according to the manufacturer’s
guidelines. Cycle sequencing reactions were carried out in both the forward and reverse
directions in an ABI 9700 thermal cycler using the ABI Big-Dye Terminator mix (v. 3.0;
Applied Biosystems, Foster City, California, USA). Forward and reverse COI sequences from
the same individual were aligned using AlignIR (v. 2.0, LI-COR Biosciences Inc., Lincoln,
Nebraska, USA). COI sequences that we generated from each cricket species were submitted to
GenBank (accession numbers JF411599-JF411602).
To design Ceuthophilus-specific primers, GenBank was searched for additional
sequences from species of Rhaphidophoridae whose COI sequences would align with the COI
region sequenced above (C. gracilipes [AY793593], Diestrammena unicolor [EF540833 ],
Dolichopoda geniculata [AY507609 ], D. laetitiae [AY793611 ], D. schiavazzii [AY507629 ],
Talitropsis crassicruris [AF320676 ], T. megatibia [AF320678 ], T. sedilloti [AF320681] were
accessed). These sequences were aligned with the sequences we generated (26 C. gracilipes, 24
C. guttulosus, 24 C. pallidipes and 5 E. puteanus). Multiple sequence alignments were done
using CLUSTAL X (Larkin et al., 2007). This alignment was used to design a primer pair
(Cpall-COI-F [5'-TGGTGATGACCAAATTTATAACG-3'] and Cpall-COI-R[5'GGAAAGCTATATCAGGAGCTCCAA-3']) that amplify a 140 bp fragment of C. pallidipes
COI for the purpose of detecting cricket DNA from mouse fecal samples.
For each mouse, the fecal pellets were pooled from each trap session, homogenized, and
washed with 95% ethanol and molecular water in preparation for extraction. Samples were
extracted using the QiaAmp DNA stool mini kit following the protocol for the “Isolation of
DNA from Stool for Pathogen Detection” (QiaAmp handbook). PCR reactions (25 μL) consisted
of 1X QIAGEN PCR buffer, 0.2 mM of each dNTP, 0.5 mM of each primer, 0.625 U QIAGEN
HotStarTaq Plus and template DNA (1 μL of total DNA). The PCR cycling protocols were 94 °C
for 1 min followed by 50 cycles of 94 °C for 50 s, 65 °C for 45 s, 72 °C for 45 s. Reaction
success was determined by electrophoresis of 10 μL of PCR product in 3% SeaKem agarose
stained with ethidium bromide (as above). Positive controls containing C. pallidipes DNA and
negative controls were included in each PCR. To test primer specificity, these primers were
screened against extractions of 82 non-target insect (including two other Ceuthophilus spp.),
mollusc and nematode species (after Chapman et al., 2010; Jaramillo et al., 2010).
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