Methods in Anopheles Research - MR4

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Chapter 6 : Dissection Techniques6.8 A. gambiae s.l. Ovarian Polytene Chromosome PreparationPage 1 of 66.8 A. gambiae s.l. Ovarian Polytene Chromosome PreparationAnthony Cornel 1IntroductionThe highest quality Anopheles gambiae s.l. polytene chromosomes are prepared from nurse cells in eggsat Christophers’ III stage (Clements 1992) of ovarian development. Chromosomes of adequatepolytenization also occur in salivary gland cells of late fourth stage larvae which are useful in larvalecological studies. Anopheles gambiae s.l. has a diploid chromosome complement of 2n = 6 with one pairof heteromorphic X and Y sex chromosomes and two pairs of autosomes numbered as 2 and 3 (seesection on Anopheles Mendelian Genetics).A typical polytene chromosome squash will produce a spread of arm configurations known as X, 2R, 2L,3R and 3L (Figure 6.8.1). The entire Y and portions of the X cannot be visualized because they areheterochromatic and under-replicated. Drawings depicting the banding patterns of An. gambiae s.l.polytene chromosomes are available to identify the divisions and subdivisions and locations ofparacentric inversions (Coluzzi et al. 2002; Holt et al. 2002).Wherever possible, multiple methods have been described from which you can choose depending onlaboratory equipment and the needs and quality of chromosome preparation required. The most crucialstep to obtain excellent chromosomes is to recognize the appropriate half gravid state of the female andlate 4 th stage larvae from which to dissect. Thereafter, the most crucial steps are the tapping proceduresto spread the chromosomes into recognizable arms. It will take a little while and practice to achieve theappropriate skills, and I suggest you spend a few days learning these skills with someone who is bothfamiliar with chromosome preparation techniques and An. gambiae s.l. chromosome banding patterns.Solutions• Anesthetic (ethyl ether, triethylamine, CO 2 , -20C freezer space)• Modified Carnoy’s fixative (three parts pure (100%) ethanol and one part glacial acetic acid).Ethanol absorbs water from the atmosphere so ensure that the ethanol is pure, as water infixative compromises quality of spreads.• Sigmacote® - (Sigma-Aldrich – St. Louis MO, USA)• Propionic acid - prepare 5% and 50% in water.• 2% lacto-aceto orcein - prepare this solution by adding slowly 2% by weight of synthetic orceinpowder to a solution of 1 part glacial acetic acid and 1 part pure lactic acid under constant stirring(use a magnetic stirrer). Remove un-dissolved orcein particulates by filtering the solution throughWhatman 3MM paper. The solution can be stored indefinitely at room temperature.• Ethanol – 70%, 90% and 100% in water.Materials• Anesthetizing mosquito chamber: 45 ml Falcon® tubes, razor blade, 925 mµ mesh or smallerscreening, glue, rubber bands, dental rubber latex sheeting (Super Dam- Patterson Brand),regular household sponge, desiccator.• 4C refrigerator• -20C freezer1 Mosquito Control Research Laboratory, Department of Entomology and Center for VectorborneDiseases, University of California at Davis, Parlier, CA 93648, E-mail: cornel@uckac.edu
Chapter 6 : Dissection Techniques6.8 A. gambiae s.l. Ovarian Polytene Chromosome PreparationPage 2 of 6• 0.13- 0.17 mm thick cover glasses of dimensions 22L X 22W mm• Frosted 1mm thick microscope slides 75 x 25 mm in size.• Beaker• Aluminum foil• Forceps• Pasteur pipettes• Absorbent paper (Whatman 3MM paper)• Dissecting needles and minutien pins• Dissecting Microscope• 1.5 ml screw cap polypropylene vials• Phase contrast compound microscope with 10X, 40 or 60 X and 100X objective lenses.• Digital imaging systemFigure 6.8.1. Chromosome squash showing the five reasonably well spread chromosome arms X, R,L, 3R and 3L of An. gambiae sensu stricto. Magnification X 600 under phase contrast. T = Telomere;C = Centromere. Specimen from Eau et Foret, Cameroon 2006.
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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