Methods in Anopheles Research - MR4

Chapter 6 : Dissection Techniques6.8 A. gambiae s.l. Ovarian Polytene Chromosome PreparationPage 4 of 6mosquito proof mesh so the mosquitoes do not escape but vapor can pass through. Place theFalcon® tube chamber, with mosquitoes in, into a dessicator saturated with C0 2 gas or a spongesoaked with the anesthetizing chemical (ethyl ether or triethylamine). Wait until the mosquitoes areknocked down before dissection.Figure 6.8.3. Examples of adult females atvarious stages of ovarian development. A –too young, B at correct age and C too old.OV indicates the developing ovaries whileBL indicates undigested blood meal.Photographs taken by Marshall Johnson.6. In conditions where no anesthetizing facilities are available, such as in the field, knock out themosquitoes by shaking the cup or container they are in from side to side for about 30 seconds. Themosquitoes will be stunned by hitting against the sides, but it takes some experience to judge howvigorously to shake the container without damaging the mosquitoes.7. Removing ovaries can be done with or without a dissecting microscope depending on your eyesightand dexterity. Without a microscope - which is by far the quickest way - gently hold the head, thoraxand base of abdomen of the mosquito between the tips of your thumb and index finger. Grab the lasttwo segments of the abdomen with forceps. Gently squeeze the base of the abdomen andsimultaneously pull on the last two abdominal segments with the forceps to extract the ovaries. In thisway, only the ovaries will pull out. Immediately place the ovaries into the vial containing modifiedCarnoy’s solution. Do not let the ovaries dry out after dissecting. After each dissection place a label(write label with a pencil, not a pen) with ovaries into the vial. The remaining head and thorax can bediscarded or preserved in a separate vial for other purposes such as for DNA extraction. If preserved,label the remaining carcass with an accession number corresponding to that of the ovary.8. To remove ovaries under a microscope, pierce the side of the mosquito with a minutien pin and on aclean microscope slide pull the last two apical abdominal segments with a forceps or needle until theovaries have extruded. Immediately place the ovaries into modified Carnoy’s.9. Dissected ovaries fixed and preserved in modified Carnoy’s for more than 24 hours typically providethe best preserved and clean chromosome spreads, especially if required for in situ hybridization.Ovaries preserved in modified Carnoy’s can be stored for many months at 4ºC or for years at -20ºC.10. Placing the whole abdomen in modified Carnoy’s also produces readable spreads but thechromosomes can have a washed out appearance after longer storage due to presence of morewater in the whole abdomen versus ovaries alone. It is not recommended to fix the entire adult inModified Carnoy’s solution as then too much water enters the preservation solution.