Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.3 Determining the Sex of Anopheles Larvae and PupaePage 1 of 43.3 Determining the sex of Anopheles larvae and pupaeMR4 StaffIntroductionThere is often an experimental need to separate the sexes before they emerge e.g. in order to preserveunmated status of females, to obtain material for molecular analysis, or to determine male/female larvalratios. Anopheles spp. differ from many other mosquitoes in that there is often no easily discernabledifference in the female/male larval or pupal size or pigmentation. Some, but not all, anophelines L4females can be identified based on the generally darker color and larger size. Here we present threemethods for determining the sexes based on larval and pupal characteristics.Larval sex determination: Option 1Figure 3.3.1. Cartoon of a ‘sandwich’ slide whichworks well to position and immobilize a larva forviewing without causing injury.An early method for sexing Anopheles larvaebased on the form of the imaginal antennal lobeshas been reported (Jones 1956), but the graphicsin the manuscript can be difficult to interpret –particularly in copies. Here we offer a refinementof the method and new images developed for An.gambiae. (Note: This method is not very usefulwith An. stephensi because the imaginal disks aredifficult to see.) The best results are usuallyobtained with 2 nd day L4s as the pre-antennal lobeis almost fully formed. All observations andphotographs were made on a stereoscope, and itis important to use the dark-field setting.Constructing the viewing slide:Materials• Standard glass microscope slide• 0.3 - 0.5 mm thick plastic spacer e.g. a thin plastic laboratory ruler cut into 1 X 1.5 cm pieces. Thethickness must be selected to support a coverslip over the gap so that a larva is held firmly butnot crushed. A stack of plastic coverglasses may be stacked and glued together to obtain theappropriate thickness.• Epoxy glueConstruction and use1. Clean the slide with ethanol and dry.2. Apply a small drop of epoxy glue to the plastic spacers.3. Glue the spacers onto the slide 0.8 - 1cm apart from each other and allow to cure.