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001_Karyotyping of Honeybee

This document presents a simple method for karyotyping honey bee (Apis mellifera) eggs to determine their ploidy levels. The technique involves using a hypotonic colchicine solution and various fixatives to prepare slides for microscopic examination, revealing metaphase cells from three-day-old eggs. This method can help assess egg viability and identify haploid eggs in worker cells.

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30 views3 pages

001_Karyotyping of Honeybee

This document presents a simple method for karyotyping honey bee (Apis mellifera) eggs to determine their ploidy levels. The technique involves using a hypotonic colchicine solution and various fixatives to prepare slides for microscopic examination, revealing metaphase cells from three-day-old eggs. This method can help assess egg viability and identify haploid eggs in worker cells.

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nirodha pegu
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Apidologie 41 (2010) 178–180 Available online at:


c INRA/DIB-AGIB/EDP Sciences, 2009 www.apidologie.org
DOI: 10.1051/apido/2009058
Scientific note

A scientific note on a simple method for karyotyping honey bee


(Apis mellifera) eggs*

Rute M. Brito, Benjamin P. Oldroyd

Behaviour and Genetics of Social Insects Laboratory, Macleay Building A12, School of Biological Sciences,
University of Sydney NSW 2006, Australia

Received 26 May 2009 – Revised and accepted 18 August 2009

honeybees / Apis / chromosomes / eggs

In the honey bee (Apis) sex is determined by the from worker cells on three consecutive days after
complementary sex determining locus or csd (Beye oviposition and 12 three day-old eggs from drone
et al., 2003), located on linkage group 3 of the cells. We cut freshly harvested eggs in half and
honey bee genome (Solignac et al., 2007). Diploid incubated them in a hypotonic colchicine solution
individuals that are heterozygous at the csd are (0.005% colchicine in 1% sodium citrate solution)
female, whereas individuals that are haploid, and on a clean glass slide for 40 minutes. We then
therefore hemizygous at the csd are male (Cook and drained the solution off the slide and saturated the
Crozier, 1995). Rarely, a diploid individual will be tissue with freshly-prepared fixative solution I (60%
homozygous at the csd, usually due to inbreeding. 1:1 acetic-ethanol: glacial acetic acid 3 mL/ethanol
These individuals develop as diploid males, but are (99.5%) 3 mL/distilled water 4 mL). We then disso-
killed by workers at the first larval instar (Woyke, ciated the tissue with two needles and quickly added
1963). Diploid males can be reared artificially, but two drops of fixative II (1:1 acetic-ethanol: glacial
doing so is tedious (Polaczek et al., 2000). acetic acid 2 mL/ethanol (99.5%) 2 mL; freshly pre-
Sometimes it is desirable to be able to deter- pared) over the spread, draining off the fixative I and
mine the ploidy of honey bee eggs. For example, blotting it from the edge of the slide using strips of
it may be useful to determine if low egg viability is filter paper. Fixative III (100% glacial acetic acid)
due to the presence of haploid eggs in worker cells, was then dripped over the preparation while drain-
or to other causes such as policing of worker-laid ing off the remaining fixative II, blotting it from
eggs or disease. Although there are effective proto- the edge of the slide. We then air dried the slides
cols for examining metaphase chromosomes from overnight and stained them the following day with
the gonads of adult queens and drones (Hoshiba freshly prepared Giemsa solution (3% in Sorensen’s
and Kusanagi, 1978; Hoshiba and Okada, 1986) or phosphate buffer pH 6.8) for 15 minutes at room
from the brain ganglia of pre-pupae (Stanimirovic temperature.
et al., 2005) we have been unable to find a procedure Slides were examined under light microscope
suitable for examining metaphase chromosomes in and photographed using a Zeiss Axiophot pho-
eggs. Here we report a simple protocol for deter- tomicroscope coupled with Olympus DP71 colour
mining the ploidy of honey bee eggs based on Imai camera.
et al. (1988), who provide elaborate details of the Preparations of day-old eggs did not reveal any
fixation procedure. cells suitable for cytogenetic analysis. Only a few
To obtain biological material we caged a queen nuclei could be seen around the egg’s micropile
on an empty comb comprising worker and drone and these showed features of prophase cells such
sized cells overnight. We then collected 10 eggs as loose chromatin and an absence of nucleoli
(Fig. 1A). Two- day old eggs showed typical inter-
Corresponding author: R.M. Brito, phase cells with compact chromatin and the pres-
[email protected] ence of nucleoli (Fig. 1B). Only the preparations
* Manuscript editor: Klaus Hartfelder from three-day old eggs showed cells in metaphase,

Article published by EDP Sciences


A method for karyotyping honey bee eggs 179

Figure 1. Nuclei and metaphase obtained from Apis mellifera eggs. A. One day-old; B: two day-old; C:
three day-old male metaphase (n = 16); D: three day-old worker metaphase (2n = 32).

6 per slide on average. These cells showed the ex- ACKNOWLEDGEMENTS


pected chromosome number for A. mellifera, n = 16
or 2n = 32 (Fig. 1C and D).
We thank Malcolm Ricketts for his help with
Although our technique yields a relatively small photo-microscopy. RMB is supported by a post-
number of metaphase cells per slide, it allows doctoral Fellowship (Conselho Nacional de Desen-
a rapid assessment of ploidy level in eggs. The volvimento Científico e Tecnológico - CNPq) and
technique is potentially useful for the detection Endeavour Awards Research fellowship.
of worker-laid (haploid) eggs in worker-cells and
queen cells or determining if individuals homozy-
Note scientifique sur une méthode simple pour
gous at multiple loci are diploid or haploid. Hy- établir le caryotype des œufs de l’abeille domes-
bridization techniques such as GISH or FISH with tique (Apis mellifera).
specific probes for known genes and subsequent
counting of positive hybridization spots could be
applied in two-day old eggs, as preparations from Eine wissenschaftliche Notiz über eine einfache
eggs of this age showed nuclei suitable for this Methode zur Karyotyp-Bestimmung von Eiern
purpose. der Honigbiene (Apis mellifera).
180 R.M. Brito, B.P. Oldroyd

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