Multiplex PCR-RFLP diagnostics of the Africanized honey bee (Hymenoptera: Apidae)

Abstract

Molecular identification of Africanized honey bees, Apis melliferascutellata (AHB) in the United States often involves the use of polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) on a portion of the mitochondrial DNA (mtDNA) cytochrome b gene. One problem with this technique is that the restriction enzyme site is only found in European honey bee subspecies (EHB) and not AHB. To enhance this technique, we developed a multiplex PCR-RFLP assay that can discriminate Africanized bees from European honey bees. A 485 bp region mtDNA cytochrome b gene was subjected to DNA sequencing from honey bees collected from Arkansas and Oklahoma. Identity of honey bee subspecies was confirmed using maximum likelihood and Bayesian phylogenetic analysis of DNA sequences. Based on DNA sequence variation, a PCR primer specific for AHB was designed. This marker was screened on 96 honeybee samples from Arkansas, Oklahoma, and New Mexico and results were congruent with those obtained by PCR-RLFP. Multiplex PCR-RFLP provides a more robust identification of maternally Africanized bees than PCR-RFLP alone.

Full text

939
Multiplex PCR-RFLP Diagnostics of the Africanized Honey Bee
(Hymenoptera: Apidae)
by
Allen L. Szalanski1* & Jackie A. McKern1
ABSTRACT
Molecular identication of Africanized honey bees, Apis mellifera scutellata
(AHB) in the United States oen involves the use of polymerase chain reac-
tion – restriction fragment length polymorphism (PCR-RFLP) on a portion
of the mitochondrial DNA (mtDNA) cytochrome b gene. One problem with
this technique is that the restriction enzyme site is only found in European
honey bee subspecies (EHB) and not AHB. To enhance this technique, we
developed a multiplex PCR-RFLP assay that can discriminate Africanized bees
from European honey bees. A 485 bp region mtDNA cytochrome b gene was
subjected to DNA sequencing from honey bees collected from Arkansas and
Oklahoma. Identity of honey bee subspecies was conrmed using maximum
likelihood and Bayesian phylogenetic analysis of DNA sequences. Based on
DNA sequence variation, a PCR primer specic for AHB was designed. is
marker was screened on 96 honey bee samples from Arkansas, Oklahoma, and
New Mexico and results were congruent with those obtained by PCR-RLFP.
Multiplex PCR-RFLP provides a more robust identication of maternally
Africanized bees than PCR-RFLP alone.
Keywords: Africanized honey bee, Apiculture, Molecular diagnostics
INTRODUCTION
e Africanized Honey Bee (AHB) in North America is a hybrid of one
of the several European Honey Bee subspecies (EHB), (Apis mellifera lin-
gustica Spinola, A. m. carnica Pollmann, A. m. caucasica Pollman, and A. m.
mellifer L.) and the African Honey Bee (Apis mellifera scutellata Lepeletier).
Africanized honey bees were rst detected in Texas in 1990 (Sugden & Wil-
liams 1990), and by 2006 had spread to another 8 states. e hybrid is virtu-
1Department of Entomology, University of Arkansas, Fayetteville, AR, USA 72701.
*Corresponding author. email: aszalan@uark.edu

940 Sociobiology Vol. 50, No. 3, 2007
ally indistinguishable in the eld from the common honey bee. Traditional
morphological diagnostics involving the Fast Africanized Bee Identication
System (FABIS) requires multiple bees from single collection locations that
must be in good morphological condition. Molecular diagnostics is ideal for
samples that are in poor morphological condition, and for samples which
consist of fewer than ve bees. Use of molecular genetic diagnostics is well
established for distinguishing AHB from EHB, and Pinto et al. (2003)
developed a polymerase chain reaction restriction fragment length polymor-
phism (PCR-RFLP) technique that distinguishes maternal lineages using the
mitochondrial DNA (mtDNA) cytochrome b gene. One problem with this
PCR-RFLP diagnostic is that the restriction enzyme site is only found in
EHB and not AHB. us, if there was a problem with the restriction digest
it may be possible for an EHB sample to appear as AHB. Another method,
involving PCR with species-specic primers (multiplex PCR) (Szalanski et al.
2004) can detect nucleotide variation at the PCR primer sites and provides a
diagnostic marker independent of RFLP. In this study, DNA sequence varia-
tion between EHB and AHB collected from Arkansas, Oklahoma, and New
Mexico was used to design an AHB specic PCR primer, and to develop a
multiplex PCR-RFLP assay that can discriminate AHB from EHB.
MATERIALS AND METHODS
Ninety-six honey bee samples were collected from 11 counties in Arkansas,
31 counties in Oklahoma, and 5 counties in New Mexico by the Arkansas
Plant Board, Oklahoma State University, and county extension personnel
from various locations in Arkansas, Oklahoma, and New Mexico. Voucher
specimens, preserved in 70-100% ethanol, are deposited at the Arthropod
Museum, Department of Entomology, University of Arkansas, Fayetteville,
AR, USA.
DNA was extracted from individual thoraces using the Puregene DNA
isolation kit D-5000A (Gentra, Minneapolis, MN). Extracted DNA was
resuspended in 50 µl of Tris:EDTA and stored at –20ºC. Samples were
subjected to PCR using the primers 5’-TATGTACTACCATGAGGA-
CAAATATC-3’ (Crosier et al. 1991) (designated as Apis-F) and 5’-ATTA-
CACCTCCTAATTTATTAGGAAT-3’ (Crosier et al. 1991) (designated
as Apis-R) (Crosier et al. 1991). ese two primers amplify a 485 bp region

941
Szalanski, A.L. & J.A. McKern — PCR-RFLP Diagnostics of Africanized Bees
of the mtDNA cytochrome b gene. PCR reactions were conducted using 4
µl of the extracted DNA per Szalanski et al. (2000), with a prole consist-
ing of 94ºC for 3 min followed by 35 cycles of 94ºC for 45 s, 50ºC for 45 s
and 72ºC for 45 s. Five µl of the PCR product were then digested with the
restriction enzyme Bgl II to identify them as EHB or AHB per Pinto et al.
(2003). Digests were run on a 2% agarose gel and visualized using a BioDocit
system (UVP Inc).
Twenty-six A. mellifera (12 EHB, and 14 AHB) samples from Arkansas
and Oklahoma identied as AHB or EHB using either FABIS (USDA-
ARS Tuscon, AZ) and / or PCR-RFLP (Pinto et al. 2003) were subjected
to DNA sequencing. PCR products were puried and concentrated using
Microcon-PCR Filter Units (Millipore, Bedford, MA). Samples were sent to
the University of Arkansas Medical School DNA Sequencing Facility (Little
Rock, AR) for direct sequencing in both directions using an ABI Prism 377
DNA sequencer (Foster City, CA). A total of three EHB haplotypes and
three AHB haplotypes were observed and the GenBank accession numbers
for these haplotypes are EF016643 to EF016648.
For phylogenetic analysis, DNA sequences from the six haplotypes from
this study were compared with other A. mellifera subspecies sequences
(Koulianos & Crosier 1991, Pinto et al. 2007) downloaded from GenBank.
e sequences were aligned using Clustal W (ompson et al. 1994), and
Apis cerana (GenBank EF180094) was used as the outgroup taxon. e best-
tting nucleotide substitution model was chosen according to the general time
reversible + gamma (GTR+G) model among 64 dierent models using the
ModelTest v 3.7 (Posada & Crandall 1998) and PAUP* 4.0b10 (Swoord 2001)
programs. Phylogenetic analysis was conducted using maximum parsimony
(MP) analysis with the best-tting evolutionary model as implemented in
PAUP*. Bootstrapping was performed using neighbor joining or MP (1000
replicates) to determine the reliability of the obtained topologies. Phyloge-
netic trees were also obtained using Bayesian inference with the GTR+G
model applying Bayesian Evolutionary Analysis Sampling Trees (BEAST)
soware v1.4.2 (Drummond & Rambaut 2003). For Bayesian inference, four
Markov chains run for 106 generations with a burn-in of 2x104 were used to
reconstruct the consensus tree.

942 Sociobiology Vol. 50, No. 3, 2007
Fig. 1. Phylogenetic relationship of six EHB and AHB haplotypes relative to other Apis cytochrome
b mtDNA sequences. GenBank accession numbers are provided for each taxon. Numbers at the tree
nodes indicate Bayesian posterior probabilities, and values above branches indicate bootstrap values
obtained from 1000 replicates using MP analysis.

943
Szalanski, A.L. & J.A. McKern — PCR-RFLP Diagnostics of Africanized Bees
RESULTS AND DISCUSSION
A total of three EHB haplotypes and three AHB haplotypes were observed
from the Arkansas, Oklahoma, and New Mexico samples (Fig. 1). Phyloge-
netic analysis using both MP and Bayesian analysis conrmed that the three
AHB haplotypes were A.m. scutellata, EHB haplotype 1 was A.m. lingustica,
and EHB haplotypes 2 and 3 were A. m. carnica (Fig. 1). For the design of an
AHB specic PCR primer, DNA sequences from the six haplotypes, along
with sequences of the other A. mellifera used for the phylogenetic analysis
(Fig. 1), were aligned using BioEdit (Hall 1999), and examined for nucle-
otide variation between EHB and AHB. e 110 to 132 bp region of the
DNA sequence had two nucleotide sites that were unique to AHB and this
genetic variation was used to design a forward PCR primer that was specic
for AHB (AHB-F 5’-CATTACTCTGAGGTGGGTTC-3’). e AHB-F
primer combined with the Apis-R reverse primer results in a 385 bp PCR
amplicon (Fig. 2).
Multiplex PCR with the AHB specic primer (AHB-F) and the two A.
mellifera primers (Apis-F, Apis-R) was tested for optimal annealing perfor-
mance in a 47-59°C temperature gradient with 2°C intervals. e optimal
PCR prole for the multiplex PCR reaction was 40 cycles of 94°C for 45 s,
55°C for 45 s, and 72°C for 60 s. Multiplex PCR combined with PCR-RFLP
per Pinto et al. (2003) was then conducted on 96 honey bee samples (40
AHB and 56 EHB) from Arkansas, Oklahoma, and New Mexico (Fig. 2).
All of the multiplex PCR-RFLP results matched those previously obtained
using PCR-RFLP.
Fig. 2. Two percent agarose gel of AHB multipex amplicons (485 and 385 bp), and EHB Bgl II digest
fragments (291 and 194 bp).

944 Sociobiology Vol. 50, No. 3, 2007
Multiplex PCR combined with RFLP provides a more robust method
to identify Africanized honey bee samples than just PCR-RFLP, by adding
a molecular marker that is specic for AHB. One problem with using only
PCR-RFLP is that the restriction enzyme site is only found in EHB and
not AHB. us, if there was a problem with the restriction digest it may be
possible for an EHB sample to appear as AHB resulting in a false positive
test for AHB. Multiplex PCR-RFLP is a more robust method for molecular
identication of AHB maternal lineages for regulatory purposes.
ACKNOWLEDGMENTS
We thank Ed Levi of the Arkansas Plant Board, and Richard A. Grantham
from Oklahoma State University for providing samples. We thank J.W. Aus-
tin, Texas A & M University for his review and critical comments. Research
was supported in part by the University of Arkansas, Arkansas Agricultural
Experiment Station.
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