Methods in Anopheles Research - MR4

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Chapter 2 : Anopheles Laboratory Biology and Culture2.4 Anopheles Culture2.4.4 Determining Egg Hatch RatesPage 1 of 22.4.4 Determining Egg Hatch RatesMR4 StaffIntroductionEgg hatching rates vary between stocks depending on intrinsic fertilization rates, semisterility due tocrossing type or presence of chromosomal aberrations and the methods used to handle eggs afteroviposition. Egg hatch rates >80% are typical. Counting hatched larvae is not a proxy for determining thehatch rate as mortality may occur in the L1 stage and these larvae are very difficult to detect. Typically,anopheline eggs that sink to the bottom of the pan do not hatch; however, they should be inspected andincluded in hatch rate data.Unhatched eggs fall into several classes which may be of interest to record: (1) Unmelanised eggs areoften observed but will inevitably fail to hatch; (2) Unhatched, melanised eggs in which no indication of anembryo can be seen; (3) Unhatched, melanised eggs in which a developing embryo is seen but neverhatches; (4) Unhatched, melanised eggs in which an embryo is alive but has not hatched.The last category is problematic. In some species, hatch is very synchronous, but in others –An. gambiae – it occurs over several days even when eggs are moist (Lehmann et al. 2006). We haveoften observed egg batches in which most eggs have hatched a day earlier, but the activity of countingthe eggs stimulates further hatch. These various types and timings of hatching should be taken intoaccount when collecting and interpreting hatching data.Materials• Filter paper• Wash bottle containing water• Fine probesEquipment• Stereoscope• 2-place denominator (counter)Determining hatch rates for eggs collected fromindividual females1. Assuming eggs have been collected in tubes, theseshould have been lined previously with filter paper(See Chapter 3.9). If the eggs are not all at the edges,carefully touch the center of the water. The oil on yourfingers will usually cause the eggs to move to the side.If not, tease them to the edge with a probe.2. Very slowly and smoothly slide the papers up the sideof the tube and transfer to a rigid, movable, flatsurface. A small piece of Plexiglass (10 X 20 cM) isideal for this.Figure 2.4.4.1. An unhatched (left) andhatched egg (right) from the same female,laid on the same day. It is apparent thelower one has hatched because of thedislocated operculum. Un-melanised eggsdo not hatch.3. Count the eggs in situ under a stereoscope recording hatched vs. unhatched on the denominator.You may need to prod or burst the egg with a probe to determine whether it is hatched. Typically theoperculum of hatched eggs will be dislodged somewhat (Figure 2.4.4.1). If only a sample of eggs isneeded for rate information, counting 50 eggs is sufficient. Otherwise, count all the eggs.
Chapter 2 : Anopheles Laboratory Biology and Culture2.4 Anopheles Culture2.4.4 Determining Egg Hatch RatesPage 2 of 2Determining hatch rates for en masse egg collectionsIn this case, the container from which eggs are taken may or may not be lined with filter paper. If it is, skipto step 2.1. Slide a small piece of filter paper (approx 2 X 6 cm) down the side of the tray above the eggs to becollected. When the paper begins to wet, slowly slide it behind the eggs until the paper is wellsubmerged.2. Slowly slide the paper on which the eggs are resting up the side of the tray until it is above the water.The eggs should adhere to the paper as it is raised.3. Transfer the paper to the counting board as described above and count.ReferencesLehmann T et al. (2006) Genetic contribution to variation in larval development time, adult size, andlongevity of starved adults of Anopheles gambiae. Infect Genet Evol 6:410-416
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