Methods in Anopheles Research - MR4

Chapter 2 : Anopheles Laboratory Biology and Culture2.4 Anopheles Culture2.4.6 Anopheles Larval CulturePage 3 of 6A reasonable density for most L3-4 anophelines is 1 larva per ml with the water level 0.5-1 cm in depth.When it is impractical to estimate the exact density in the early (L1-2) stages, larvae are usually culturedat a high density. For this reason, the rearing schedule (Chapter 1) is designed for thinning progressivelyin stages. Some species such as An. quadrimaculatus and An. freeborni may culture more successfully at ahigher density in the early instars than is typical for An. gambiae.FeedingAt a constant temperature and given an appropriate amount of diet, the time from hatch to pupationshould be predictable within one day from generation to generation. If pupation is delayed more than aday or two, any of the following could be responsible: temperature is too low, inadequate food was givenat some stage, the density was too high, or excessive food was given in the early stages. Poor cultureresults in disparate developmental rates leading to pupation over the course of several days. This resultsin a great deal of extra work for the technicians. Ideally, almost all pupae form over 2 to 3 days.Examine the pans daily (even when you do not feed) to ensure that the larvae are developing asexpected and the density is appropriate. If you notice great differences in sizes of larvae between orwithin one tray of a cohort at any time, you likely have them too crowded and/or they are underfed. Notsurprisingly, the amount of food provided daily must increase as the larvae develop and as their densityincreases. However, there is a limit to the amount of food and larvae you can place in one tray, so werecommend adhering initially to the density guidelines above and modifying only the amount of diet.Underfed trays will contain larvae that die, are slow-growing or are variable in their development rate. Inextreme cases, unusually long fecal pellets will be observed due, presumably, to re-ingestion of feces.L4s that are overcrowded and/or underfed will be small and have little fat body accumulation.Overfeeding is common and is indicated by numerous observations that precede larval death.1. Foul smell. If you smell a foul odor when you remove the cover, you’re feeding too much. A healthyorganic odor is normal. However, what is considered healthy is admittedly dependent on personalaesthetics!.2. Excessive turbidity. Yellowish to greenish-colored water is fine and often appears in later stages ofrearing (referred to as gelbstoff). However, if the water is turbid, feed less or not at all until the waterclarifies. If turbidity persists, filtering the larvae out from the old culture water and at least a partialwater change may be necessary. Greater turbidity is tolerable during the L3 and L4 stages whereasL1s and L2s are more sensitive. You will develop judgment regarding how much turbidity isappropriate.3. Excessive surfactants. When the water in the pan is agitated, bubbles that form should burst rapidly.If they persist, bacterioneuston has formed an excessive surface microlayer which is not healthy forAnopheles larvae. Check for these by sloshing the water gently. Larvae exposed to water with highlevels of surfactants often do not survive and re-feeding of the adult stock may be necessary. Ifbubbles persist, filtering the larvae out from the old culture water or dragging a tissue over the surfaceand at least a partial water change are recommended. If this is observed routinely, the cultureconditions must be changed.Overfeeding and microbes in the larval culture can cause irregular scum to form on the bottom of thetrays. These pans should be scrubbed with a detergent, rinsed thoroughly and autoclaved or bleached. Itis important to remember that in almost all cases, a thin layer will form on the bottom of the pan fromwaste materials and settled diet. It is a problem only when the layer becomes gelatinous or jelly-like inappearance, irregular in its reflectance, or contains bubbles. Change the container and reduce thefeeding rate.