Supplemen a y File 1: PNA Clamp De elopmen
The PNA clamp (PNA Bio, Thousand Oaks, CA) was de eloped o educe ampli ica ion
o Apis melli e a, o be used in conjunc ion wi h he COI me aba coding p ime s FwhF2
(5’-GGDACWGGWTGAACWGTWTAYCCHCC-3’) and FwhR2n (5’-
GTRATWGCHCCDGCTARWACWGG-3’) (Vamos e al. 2017). These me aba coding
p ime s we e chosen as hey ha e been shown o eco e >95% o species in mock
a h opod communi ies, show minimal a ia ion in species eco e y based on changes
o annealing empe a u e, and ampli y a sho ~200 bp egion ideal o deg aded eDNA
samples (Elb ech e al. 2019). The PNA clamp was designed o high speci ici y o A.
melli e a and mul iple nucleo ide misma ches o ele an bee pes s including Va oa
spp. and Asian honeybee species.
Con i ma ion o educed A. melli e a ampli ica ion
Tissue samples o A. melli e a, A. ce ana, A. lo ea, V. des uc o , and V. jacobsoni we e
ex ac ed using a chelex DNA ex ac ion me hod and quan i ied using a Nanod op 8000
Spec opho ome e (The mo Scien i ic). Samples we e hen dilu ed o 2 ng/µL. All
samples we e ini ially ampli ied ia a g adien quan i a i e PCR (qPCR) o he COI
ma ke s only o con i m app op ia e cycling condi ions. qPCR eac ions we e ca ied
ou in 15 µL olumes using 7.5 µL o SensiFAST SYBR No-ROX mix (Bioline), 0.6 µL each
o o wa d and e e se p ime (10 uM), 4.3 µL o ul apu e H2O, and 2 µL o DNA.
Samples we e ampli ied using a CFX96 Touch Real-Time PCR De ec ion Sys em
(BioRad) ollowing an ini ial dena u a ion a 95°C o 5 minu es, ollowed by 45 cycles o
95°C o 30 seconds, annealing s ep (45-50°C) o 30 seconds, and an ex ension o 72°C
o 50 seconds. Following he iden i ica ion o a sui able annealing empe a u e he
PNA clamp was es ed a inal eac ion concen a ions o 1, 2, 3, 4, and 5 uM o iden i y
op imal pe o mance o he clamp. qPCR eac ions and p o ocols we e ollowed as
desc ibed abo e, wi h he ollowing modi ica ions: amoun o H2O adjus ed o accoun
o he addi ion o he PNA clamp, 50°C annealing empe a u e, and he numbe o
qPCR cycles educed o 40. qPCRs we e un in iplica e o ensu e he esul s p oduced
we e consis en wi h DNA nega i e con ols un h oughou , and samples ampli ied
wi hou any PCR blocke s in each eac ion as an addi ional con ol.
The PNA clamp showed a educ ion in ampli ica ion o A. melli e a by six cycles, while
only impac ing V. des uc o ampli ica ion by one cycle (Fig.1). No o he non- a ge
species es ed showed any educed ampli ica ion when he PNA clamp was included in
he PCR eac ion. The e ec i eness o he PNA clamp did no conside ably di e ac oss
any o he concen a ions es ed; we elec ed o use a 2uM concen a ion o
me aba coding samples o his s udy.
Fig. 1 Mean Cq esul s ac oss h ee eplica e uns, es ed on h ee honeybee species (A. melli e a, A.
ce ana, and A. lo ea) and wo a oa mi e species (V. des uc o and V. jacobsoni), a a ange o PNA
clamp concen a ions. Shaded egions ep esen s anda d de ia ions.
Re e ences
Elb ech V, B aukmann TWA, I ano a NV, P osse SWJ, Hajibabaei M, W igh M, Zakha o EV, Hebe
PDN, S einke D (2019) Valida ion o COI me aba coding p ime s o e es ial a h opods. Pee J
7: e7745. h ps://doi.o g/10.7717/pee j.7745
Vamos E, Elb ech V, Leese F (2017) Sho COI ma ke s o eshwa e mac oin e eb a e me aba coding.
Me aba coding and Me agenomics 1: e14625. h ps://doi.o g/10.3897/mbmg.1.14625
