Chapter 2 : Anopheles Laboratory Biology and Culture 2.4 Anopheles Culture 2.4.

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2.4.5 Separating Larvae and Pupae

MR4 Staff Introduction Four mosquito features used alone or in combination can 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, adults will 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 not work equally well for all species and must be tested. While pipetting will select pupae of all species based on appearance, the most uniformly effective en masse method for all species is size separation using a glass plate pupa separator (Fay and Morlan 1959). Another device based on the size differences is that of McCray (McCray 1961) but this does not allow on-the-fly adjustment as readily as does the Fay and Morlan device. Separate by appearance The simplest, but most time consuming method involves using a pupa picker to individually manually remove pupae from the rearing pan and place them into an emergence cup. This method is appropriate only when small numbers of pupae are present. Examples of some utensils are shown in Figure 2.4.5.1. Figure 2.4.5.1. Examples of utensils useful for manual pupae separation or hand picking. On the left is a piece of wire mesh glued to a metal spatula and is used to scoop pupae. The middle implement is made of a 1,000 l disposable pipette tip, the tip of which has been trimmed off and fitted with a 2 ml pipette bulb. The pupa picker on the right is a disposable pipette especially good for avoiding contamination as it is transparent. Rinsing these in hot water often is necessary to prevent contamination.

Separation by differential buoyancy of pupae and larvae Anopheles larvae are negatively buoyant whereas pupae are positively buoyant. Pupal and larval swimming activity counteracts this useful difference, but chilled water can be used to reduce this. Swirling One quick method is swirling larvae and pupae in a cup: larvae will accumulate in the middle on the bottom and the pupae at the surface and sides of the cup. Using a pipette such as one shown in Figure 2.4.5.1, remove the larvae from the middle or the pupae from the sides.

Chapter 2 : Anopheles Laboratory Biology and Culture 2.4 Anopheles Culture 2.4.5 Separating Larvae and Pupae v 1 Page 2 of 4 Swirling with chilled water A variation is to use ice water to stun the mosquitoes allowing them to be separated in water without their behavior interfering (Weathersby 1963). 1. Collect the pupae and larvae together in a strainer and wash them into a small bowl (Figure 2.4.5.2). 2. Add ice-cold water free of ice (Figure 2.4.5.3). 3. Quickly swirl and wait until separation occurs (Figure 2.4.5.4-5). 4. Gently pour the pupae into a sieve and transfer to emergence cups containing culture temperature water. Notes A separatory funnel or Imhoff cone can also be used with the ice water method (Figure 2.4.5.6) (Hazard 1967). This is a good method for large numbers of a single strain. Ice water increases larval mortality in some strains/species so the time of ice water exposure should be minimized. Larvae typically only survive this treatment 2 or 3 times, so this is most useful when large numbers are cultured and picking is limited to 2 to 3 days. Carbon dioxide anesthesia has also been used for the purpose of immobilizing pupae and larvae for separation, though we have not tried it (Lin and Georghiou 1976). This method could be similar to ice water in its usefulness.

Figure 2.4.5.2. Place pupae and larvae in a small volume of water.

Figure 2.4.5.3. Pour in ice water leaving enough room for swirling and pouring.

Figure 2.4.5.6. Chilled water method as shown to left but using an Imhoff cone. Larvae are on the bottom and pupae at the top of the funnel.

Figure 2.4.5.4. Swirl gently and then allow separating. Larva will accumulate in the bottom middle of the cup and pupae will float at the top and around the sides.

Figure 2.4.5.5. Side view of larval/pupal separation in ice water.

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Separation by larger size of pupae Fay-Morlan Separator A custom-built glass-plate pupa separator (Figure 2.4.5.7) is good for large numbers of one strain and has minimal harmful effects. Two roughly parallel glass plates are adjusted with knobs to control the spacing of the upper and lower portions of the glass. By controlling the space you can trap the pupae (larger diameter) and allow the larvae (smaller) to flow through into a tray. Then the glass plates are loosened and the pupae flushed into a separate pan. This method can also be used to separate the sexes of pupae when male and female size differs considerably though it is not usually possible with Anopheles mosquitoes since both sexes are roughly the same size in the pupal stage (see Chapter 2, Physiology section). Care must be taken not to overload the device or it will become congested with pupae and the flow-through of larvae will be prevented. The risk of contamination is the main drawback to this method as larvae and pupae may get caught on the edges and be inadvertently transferred to another stock. Rinsing thoroughly between strains can prevent such transfer.

Figure 2.4.5.7. Custom-built pupa separator relying on the size difference between pupa and larva for separation (produced by the John Hock Company).

Figure 2.4.5.8. The McCray gate separator. The closest gate to the viewer has a narrower gap than the farthest gate; this is set up to separate dimorphic pupae and larvae of Aedes aegypti.

Gate Separator A smaller machine is the McCray Separator (McCray 1961). The separator shown utilizes a series of pre-cut aluminum plates that can be adjusted to catch pupae of varying sizes; however these cannot be adjusted during use - only between (Figure 2.4.5.8). It is especially good for separating pupae that have distinct size dimorphism such as Ae. aegypti. The contents of the larval rearing pans are poured into the upper chamber of the sluice where the plate gap is set to catch the largest pupae first. By applying a gentle stream of water, the smaller size pupae and larvae are washed down to the lower chamber which is set at a slightly narrower gap to catch any smaller pupae. From here, the larvae are flushed down into a new rearing pan where they can be fed and left to pupate. After all the larvae have been rinsed through, remove the aluminum dividers and rinse pupae into a new pan and transfer to a cage for emergence. As with the Fay-Morlan Separator, care must be made to ensure that all the pupae have been washed from the apparatus to avoid contamination when handling several different stocks.

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Gentle vortex: Separate by both activity and buoyancy The vortex method not using chilled water ranges from great to poor depending on the mosquito strain. A variation is to collect pupae and larvae in a strainer and washing them into a Florence flask with fresh water (Figure 2.4.5.9). Swirl as you fill to the top. Most of the larvae will dive and most of the pupae will rise to the neck. When separation is maximal, pour the pupae off. This method is seldom 100% effective, so some hand-picking is required. This can also work with an Imhoff funnel (as shown in Figure 2.4.5.6) in which case the larvae are drained from the bottom. Cool water will improve the separation, but take care that repeated chilling is avoided as much as possible to prevent mortality.

Figure 2.4.5.9. An. gambiae larvae and pupae in the process of separation based on activity and buoyancy. Pupae are floating to the surface.

References Fay RW, Morlan HB (1959) A mechanical device for separating the developmental stages, sexes, and species of mosquitoes. Mosq News 19 144-147 Hazard EI (1967) Modification of the ice water method for harvesting Anopheles and Culex pupae. Mosq News 27 115-116 Lin CS, Georghiou GP (1976) Tolerance of mosquito larvae and pupae to carbon dioxide anesthesia. Mosq News 36:460-461 McCray EM (1961) A mechanical device for the rapid sexing of Aedes aegypti pupae. J Econ Entomol 54 819 Weathersby AB (1963) Harvesting mosquito pupae with cold water. Mosq News L1 - 2927pdf 23:249 251