Methods in Anopheles Research - MR4

Chapter 2 : Anopheles Laboratory Biology and Culture2.4 Anopheles Culture2.4.7 Separating Larvae and PupaePage 1 of 42.4.7 Separating Larvae and PupaeMR4 StaffIntroductionFour features used alone or in combination to separate pupa from larva are: 1) buoyancy 2) size 3)activity and 4) appearance. Pupa of most species should be separated from larva daily; otherwise, adultswill emerge. Some species require a longer pupal development and can be separated every other day.There are several larva/pupa separation methods that use equipment varying in sophistication. All will notwork equally well for all species and must be tested. While pipetting will select pupae of all species basedon appearance, the most uniformly effective en masse method for all species is size separation using aglass plate pupa separator (Fay and Morlan 1959). Another device based on the size differences is that ofMcCray (1961) but this does not allow on-the-fly adjustment as readily as does the Fay and Morlandevice.Separate by appearanceThe simplest, but most time consuming method involves using a “pupa picker” to individually manuallyremove pupae from the rearing pan and place them into an emergence cup. This method is appropriateonly when small numbers of pupae are present. Examples of some utensils are shown in Figure 2.4.7.1.Figure 2.4.7.1. Examples of utensilsuseful for manual pupae separation or“hand picking”. On the left is a piece ofwire mesh glued to a metal spatula and isused to scoop pupae. The middleimplement is made of a 1,000 µldisposable pipette tip, the tip of which hasbeen trimmed off and fitted with a 2 mlpipette bulb. The pupa picker on the rightis a disposable pipette especially good foravoiding contamination as it istransparent. Rinsing these in hot wateroften is necessary to preventcontamination.Separation by differential buoyancy of pupae and larvaeLarvae are negatively buoyant whereas pupae are positively buoyant. Pupal and larval activitycounteracts this useful difference, but chilled water can be used to reduce this.SwirlingOne quick method to try is swirling larvae and pupae a cup: larvae will accumulate in the middle on thebottom and the pupae at the sides of the cup. Using a pipette such as one shown in Figure 2.4.7.1,remove the larvae from the middle or the pupae from the sides.