Rapid method for DNA extraction from the honey bee Apis mellifera and the parasitic bee mite Varroa destructor using lysis buffer and proteinase K.

We developed a rapid method for extraction of DNA from honey bees, Apis mellifera, and from the parasitic bee mite, Varroa destructor. The advantages include fast processing and low toxicity of the substances that are utilized. We used lysis buffer with nonionic detergents to lyse cell walls and proteinase K to digest proteins. We tested whole thorax, thoracic muscle mass, legs, and antennae from individual bees; the mites were processed whole (1 mite/sample). Each thorax was incubated whole, without cutting, because exocuticle color pigment darkened the extraction solution, interfering with PCR results. The procedure was performed with autoclaved equipment and laboratory gloves. For each sample, we used 100 µL lysis buffer (2 mL stock solution of 0.5 M Tris/HCl, pH 8.5, 10 mL stock solution of 2 M KCl, 500 µL solution of 1 M MgCl2, 2 mL NP40, and 27.6 g sucrose, completed to 200 mL with bidistilled water and autoclaved) and 2 µL proteinase K (10 mg/mL in bidistilled water previously autoclaved, as proteinase K cannot be autoclaved). Tissues were incubated in the solutions for 1-2 h in a water bath (62°-68 °C) or overnight at 37 °C. After incubation, the tissues were removed from the extraction solution (lysis buffer + proteinase K) and the solution heated to 92 °C for 10 min, for proteinase K inactivation. Then, the solution with the extracted DNA was stored in a refrigerator (4°-8 °C) or a freezer (-20 °C). This method does not require centrifugation or phenol/chloroform extraction. The reduced number of steps allowed us to sample many individuals/day. Whole mites and bee antennae were the most rapidly processed. All bee tissues gave the same quality DNA. This method, even using a single bee antenna or a single mite, was adequate for extraction and analysis of bee genomic and mitochondrial DNA and mite genomic DNA.

Morphometric and genetic changes in a population of Apis mellifera after 34 years of Africanization.

Though the replacement of European bees by Africanized honey bees in tropical America has attracted considerable attention, little is known about the temporal changes in morphological and genetic characteristics in these bee populations. We examined the changes in the morphometric and genetic profiles of an Africanized honey bee population collected near where the original African swarms escaped, after 34 years of Africanization. Workers from colonies sampled in 1968 and in 2002 were morphometrically analyzed using relative warps analysis and an Automatic Bee Identification System (ABIS). All the colonies had their mitochondrial DNA identified. The subspecies that mixed to form the Africanized honey bees were used as a comparison for the morphometric analysis. The two morphometric approaches showed great similarity of Africanized bees with the African subspecies, Apis mellifera scutellata, corroborating with other markers. We also found the population of 1968 to have the pattern of wing venation to be more similar to A. m. scutellata than the current population. The mitochondrial DNA of European origin, which was very common in the 1968 population, was not found in the current population, indicating selective pressure replacing the European with the African genome in this tropical region. Both morphometric methodologies were very effective in discriminating the A. mellifera groups; the non-linear analysis of ABIS was the most successful in identifying the bees, with more than 94% correct classifications.

The Apis mellifera pupal melanization program is affected by treatment with a juvenile hormone analogue.

Apis mellifera treated during different developmental phases with pyriproxyfen, a juvenile hormone analogue, show profound alterations in cuticular pigmentation and sclerotization. When the treatment is effected during the feeding phase of the fifth larval instar (LF5), the pupal development is blocked and pigmentation does not occur. Treatment of older larvae, at the spinning phase of the fifth larval instar (LS5), of prepupae (PP) or pupae at the beginning of the pupal period (Pw, white-eyed, unpigmented cuticle pupae) does not impair pigmentation, but, instead, this process is accelerated, intensified and abnormal. Hormonal treatment during these developmental phases (LS5, PP and Pw) induces earlier activity of phenoloxidase, an enzyme of the reaction chain leading to melanin synthesis. Treated pupae have significantly higher enzymatic levels and show a graded response in phenoloxidase activity after treatment with 0.1, 1 or 5&mgr;g pyriproxyfen. Besides pigmentation, other developmental events were also altered in treated bees: pupal development was shortened, and the expression of esterase-6 activity, the onset of which coincides with the beginning of pigmentation, was shifted with the precocious initiation of this process in treated pupae. The significance of these results is discussed in relation to the mode of hormonal action on cuticular pigmentation in insects.