DOI: 10.13102/sociobiology.v61i2.234-236Sociobiology 61(2): 218-220 (June, 2014) Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 Assessing the Utility of a PCR Diagnostics Marker for the Identification of Africanized Honey Bee, Apis mellifera L., (Hymenoptera: Apidae) in the United States AL Szalanski, AD Tripodi The Africanized honey bee was first detected in Texas in 1990 (Sugden & Williams, 1990), and USDA reports it has been established in ten states: Arizona, Arkansas, Cali- fornia Florida, Louisiana, Nevada, New Mexico, Oklahoma, Texas and Utah (Anonymous, 2011) Following a tragic death in 2010, Africanized honey bees were confirmed in Georgia (Berry, 2011). Hybrids between Africanized and European honey bees are morphologically similar and difficult to dis- tinguish from one another. Two kinds of laboratory-based techniques, morphometric analysis (Rinderer et al., 1993) and molecular diagnostics (Sheppard & Smith, 2000), are commonly employed to determine Africanized status, but to our knowledge, these have not been compared. The typical morphometric approach to Africanized honey bee detection is the Fast Africanized Bee Identification System (FABIS) method (Rinderer et al., 1987), although more precise methods such as Automatic Bee Identification System (ABIS) (Steinhage et al. 2001) are now available. Morphometric diagnostic techniques require measurements from 10 or more freshly collected specimens (Meixner et al., 2013) to assign a colony to one of four categories: 1) Africanized (AHB), 2) AHB with European (EHB) traits, 3) EHB with AHB traits and 4) EHB (Sylvester & Rinderer Abstract An assessment of a molecular diagnostic technique for distinguishing Africanized hon- ey bees from European honey bees in the United States was conducted. Results from multiplex PCR diagnostics of a mitochondrial DNA cyt-b marker corresponded with re- sults based on COI-COII sequencing analysis, but differed from morphometric analysis results. We suggest utilizing both multiplex PCR and morphometric methods for Afri- canized honey bee diagnostics in the United States, when possible. Sociobiology An international journal on social insects University of Arkansas, Fayetteville, Arkansas, USA. Article History Edited by Gilberto M M Santos, UEFS - Brazil Received 17 December 2013 Initial acceptance 20 February 2014 Final acceptance 05 March 2014 Keywords Apis mellifera, molecular diagnostics, Africanized honey bee, mtDNA, USA Corresponding author Allen Szalanski Dept. Entomology, University of Ar- kansas, 319 Agriculture Building Fayetteville, AR 72701 E-Mail: aszalan@uark.edu 1987). Molecular diagnostics rely upon mitochondrial DNA (mtDNA), typically a region of the cytochrome b gene (Pinto et al. 2003). Identification of Africanized bees with mtDNA is relatively easy, because a single worker in any condition can represent a colony’s mtDNA lineage (Sheppard & Smith, 2000). Yet, since mtDNA is maternally inherited, mtDNA markers cannot determine if a European queen has mated with Africanized drones, and such colonies will remain un- detected with mtDNA-based techniques. Molecular identifi- cation of Africanized bees is also conducted with an mtDNA cytochrome b marker (cyt-b) as it has a low level of intraspe- cific variation in honey bees (Crozier et al., 1991; Szalanski & McKern, 2007). Techniques typically used are either PCR- RFLP (Pinto et al., 2003) or multiplex PCR using a primer specific for Africanized honey bees (Szalanski & McKern, 2007). Both of these methods yield identical results. Despite the utility of molecular markers, it is unknown how well they correlate with morphometric identification and how the cyt-b marker can distinguish the A (African) lineage of honey bees relative to the O (Middle Eastern), C (Eastern Europe) and M (Western Europe) lineages (Ruttner, 1987), which are often determined through COI-COII sequencing. The objective of this study was to determine how a cyt-b multiplex technique SHORT NOTE Sociobiology 61(2): 234-236 (June, 2014) 235 for distinguishing Africanized from European bees compares with COI-COII DNA sequence and FABIS morphometric techniques. A total of 968 samples from swarms, feral colonies and managed honey bee colonies collected from Arizona, Arkansas, California, Florida, Georgia, Hawaii, Kansas, Louisiana, Missouri, Mississippi, Nebraska, New Mexico, Oklahoma, Texas and Utah during 1991-2013 were analyzed. Samples were collected by various agencies and preserved in 70-100% ethanol. Samples collected from Texas from 1991 to 2008 were identified as Africanized (AHB, n = 54), AHB with evidence of introgression of European genes (AHB.E, n = 23), EHB with evidence of Africanized genes (EHB.A, n = 22) or EHB (EHB, n = 39) using FABIS following Rinderer et al. (1993). For the multiplex PCR diagnostics, DNA was extract- ed from two workers from each sampled colony, and PCR of a portion of the cyt-b region was conducted per Szalan- ski and McKern (2007). This multiplex results in a control amplicon of 485 bp for both Africanized and European bees and a 385 bp amplicon unique to Africanized bees. Samples exhibiting a single electrophoretic band are diagnosed as Eu- ropean (multiplex EHB) and those with two are diagnosed as Africanized (multiplex AHB). DNA sequencing analysis of a COI-COII marker was conducted following Szalanski and Magnus (2010), and samples from that study were addi- tionally subjected to our multiplex procedure for comparison (n = 360). DNA sequences were identified to haplotype and assigned to lineage using GenBank BLAST searches and our own database. Africanization diagnoses from cyt-b multiplex diagnostics, COI-COII DNA sequencing and FABIS mor- phometric analysis were then compared with one another. Of the 968 samples subjected to multiplex diagnos- tics, 318 samples were diagnosed as Africanized and 650 were diagnosed as European (Table S1). A total of 12 haplo- types from the A (African) lineage were observed, and four O (Middle Eastern), five M (Western Europe) and 11 C (Eastern Europe) haplotypes were compared with the multiplex results (Table 2). All of the multiplex PCR identified Africanized samples fell within the A lineage while the European samples were O, M or C lineages. The multiplex method reaches ex- actly the same diagnoses as the sequencing method but can be carried out in a single PCR step without subsequent DNA sequencing. Africanization diagnoses in the multiplex and FABIS methods differed quite dramatically (Table 3). Of the 107 samples the exhibiting Africanized signature in the PCR-mul- tiplex, only 84 were diagnosed as of African descent (AHB, Table 1. Sampled states and results of multiplex PCR identification of samples as Africanized or European honey bees. State N Multiplex AHB Multiplex EHB Arizona 1 0 1 Arkansas 143 1 142 California 3 3 0 Florida 1 1 0 Georgia 2 1 1 Hawaii 124 1 123 Kansas 18 0 18 Louisiana 24 0 24 Mississippi 20 0 20 Missouri 25 0 25 Nebraska 19 0 19 New Mexico 62 45 17 Oklahoma 178 58 120 Texas 140 109 31 Utah 208 99 109 Total 968 318 650 Table 2. Correlation of mitochondrial DNA COI-COII DNA se- quence lineages with cyt-b multiplex PCR identification. Lineage (n haplotypes) N Multiplex AHB Multiplex EHB C (11) 173 0 173 M (5) 12 0 12 O (4) 19 0 19 A (12) 156 156 0 Total 360 156 204 AHB.E and EHB.A) using FABIS. This suggests that 21% of Africanized bees in our sample were cryptically Africanized and undetectable using morphology-based methods. Similar- ly, of the 99 bees exhibiting morphometric characteristics of Africanization (AHB, AHB.E and EHB.A), 15% were mis- diagnosed as European by the multiplex. Interestingly, our results suggest that only a small proportion (15%) of colonies that exhibit Africanized morphology were founded by Euro- pean queens inseminated by Africanized drones. COI-COII lineage data includes haplotype determina- tions from Szalanski and Magnus (2010). This study verified the utility of using a cyt-b PCR- multiplex technique for identifying honey bees of the A lineage in the United States. Identification of Africanized matrilines through cytb-b diagnosis parallels classic COI- COII matriline determination through sequencing, but in a single-step, lesser cost procedure. We also found evidence that using either FABIS or mtDNA determination alone may underestimate the occurrence of Africanized populations. For greater certainty in diagnosing Africanized populations of honey bees, we suggest utilizing both morphological and molecular methods when the number of samples makes this possible. AL Szalanski & AD Tripodi - Africanized honey bee diagnostics236 Acknowledgements We thank numerous beekeepers and the Utah Depart- ment of Agriculture and Food, along with Ed Levi, Richard Grantham, Danielle Downey, Lisa Bradley and Clarence Collison for providing samples. We thank Lisa Bradley for doing the morphometric analysis of the Texas samples, and Clinton Trammel for assisting with the genetic analysis. This research was supported in part by the University of Arkansas, Arkansas Agricultural Experiment Station. References Anonymous (2011). Map of the spread of Africanized honey bee by year. http://www.ars.usda.gov/research/docs. htm?docid=11059&page=6. 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