Browsing by Author "Sheppard, Walter S."
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- Evidence of African honey bees mitotypes in the southern United States prior to Africanization as revealed by mtDNA sequence dataPublication . Pinto, M. Alice; Sheppard, Walter S.; Johnston, J. Spencer; Rubink, William L.; Coulson, Robert N.; Patton, John C.The standard protocol for assessing honey bees with a mitotype derived from an African origin is the amplification of a segment of cytochrome b and subsequent digestion of the amplified fragment with the restriction enzyme Bgl II. A previous survey of 451pre-Africanization honey bees from the southern U. S. revealed three bees with restriction pattern consistent with an African mitotype. To confirm these bees truly represented mitotypes of African origin we developed a new primer pair for amplification of cytochrome b, which utilizes internal sequencing primers to allow high quality direct sequence products. Using this system we amplified a mtDNA fragment of ~1,200 base-pairs (bp), taht included most of cytochrome b, serine (UCA) tRNA, and a small portion (s) of the ND-1 gene (s). Honey bees from ten morphometrically identified Apis mellifera subspecies (42 honey bee workers, each representing a different colony) from Old World and three honey bee workers (each representing a different colony) from the southern United States exhibiting an African phenotype as revealed by BglII restriction enzyme analysis were sequenced. The analysis showed that two of the three honey bees were of eastern European ancestry. These bees had lost the BglII cut site by a first position (C->A) transversion mutation. The third honey bee was found to have a sequence of African clade bees. This defining substitution for the African clade was found to be a third position (T->C) transiton mutation.
- Hinf-I digestion of cytochrome oxidase I region is not a diagnostic test for A-m. lamarckiiPublication . Kandemir, Irfan; Pinto, M. Alice; Meixner, Marina D.; Sheppard, Walter S.Restriction fragment length polymorphism of whole mitochondrial DNA or PCR amplified mtDNA regions are known to be useful in discriminating among honey bee lineages and also some individual subspecies. In this study, PCR-amplified fragments of cytochrome oxidase I (CO-I) and cytochrome B (Cyt B) of honey bees sampled from different countries (Cyprus, Turkey, Ethiopia, Syria and Egypt) were digested with Hinf I and Bgl II restriction enzymes, respectively. Eastern Europe and Mediterranean honey bee subspecies were separated by the Cyt B/Bgl II analysis, although Hinf I digestion of the CO-I region yielded much finer resolution within different honey bee lineages. Here we report that CO-I/Hinf-I is a discriminative test for the mitochondrial “O” lineage, rather than a diagnostic site for A. m. lamarckii.
- Honey bees (Hymenoptera: Apidae) of african origin exist in non-africanized areas of the Southern United States: evidence from mitochondrial DNAPublication . Pinto, M. Alice; Sheppard, Walter S.; Johnston, J. Spencer; Rubink, William L.; Coulson, Robert N.; Schiff, Nathan M.; Kandemir, Irfan; Patton, John C.Descendents of Apis mellifera scutellata Lepeletier (Hymenoptera: Apidae) (the Africanized honey bee) arrived in the United States in 1990. Whether this was the Þrst introduction is uncertain. A survey of feral honey bees from non-Africanized areas of the southern United States revealed three colonies (from Georgia, Texas, and New Mexico) with a diagnostic African mitochondrial DNA cytochrome b/BglII fragment pattern. To assess maternal origin of these colonies, we developed a primer pair for ampliÞcation of a cytochrome b fragment and sequenced using internal sequencing primers. Samples of the three reported honey bee colonies plus another 42 representing the 10 subspecies known to have been introduced in the United States were sequenced. Of the three colonies, the colonies from Texas and New Mexico matched subspecies of European maternal ancestry, whereas the colony from Georgia was of African ancestry. Contrary to expectations, the mitotype of the latter colony was more similar to that exhibited by sub-Saharan A. m. scutellata than to the mitotypes common in north African A. m. intermissa Maa or Portuguese and Spanish A. m. iberiensis Engel. This Þnding was consistent with anecdotal evidence that A. m. scutellata has been sporadically introduced into the United States before the arrival of the Africanized honey bee from South America.
- Identification of africanized honey bee (Hymenoptera: apidae) mitochondrial DNA: validation of a rapid polymerase chain reaction-based assayPublication . Pinto, M. Alice; Johnston, J. Spencer; Rubink, William L.; Coulson, Robert N.; Patton, John C.; Sheppard, Walter S.Polymerase chain reaction (PCR)-ampliÞed mitochondrialDNA(mtDNA)assays have been used in studies of the Africanization process in neotropical feral and managed honey bee populations. The approach has been adopted, in conjunction with morphometric analysis, to identify Africanized bees for regulatory purposes in the United States such as in California. In this study, 211 Old World colonies, representing all known introduced subspecies in the United States, and 451 colonies from non-Africanized areas of the southern United States were screened to validate a rapid PCR-based assay for identiÞcation of Africanized honey bee mtDNA. This PCR-based assay requires a single enzyme digestion (BglII) of a single PCR-ampliÞed segment of the cytochrome b gene. The BglII polymorphism discriminates the mitochondrial haplotype (mitotype) of Apis mellifera scutellata L. (ancestor of Africanized bees) from that of A. m. mellifera, A. m. caucasia, A. m. ligustica, A. m. carnica, A. m. lamarcki, A. m. cypria, A. m. syriaca, and some A. m. iberiensis, but not from that of A. m. intermissa and some A. m. iberiensis. Nonetheless, given the very low frequency ( 1%) of African non-A. m. scutellata mitotype present before arrival of Africanized bees in the United States, cytochrome b/BglII assay can be used to identify maternally Africanized bees with a high degree of reliability.