Accelerating the Morphogenetic Cycle of the Viral Vector Aedes aegypti Larvae for Faster Larvicidal Bioassays

Abstract

Any bioassay to test new chemically synthesized larvicides or phytolarvicides against Culicidae and more harmful mosquito species, such as Aedes aegypti and Aedes albopictus, which specifically transmit dengue, yellow fever, chikungunya viral fevers as well as Zika virus, or Anopheles gambiae, a vector for malaria and philariasis, requires thousands of well-developed larvae, preferably at the fourth instar stage. The natural morphogenetic cycle of Aedes spp., in the field or in the laboratory, may extend to 19 days at room temperature (e.g., 25°C) from the first permanent contact between viable eggs and water and the last stage of larval growth or metamorphosis into flying adults. Thus, accelerated sequential molting is desirable for swifter bioassays of larvicides. We achieved this goal in Aedes aegypti with very limited strategic and low-cost additions to food, such as coconut water, milk or its casein, yeast extract, and to a lesser extent, glycerol. The naturally rich coconut water was excellent for quickly attaining the population of instar IV larvae, the most advanced one before pupation, saving about a week, for subsequent larvicidal bioassays. Diluted milk, as another food source, allowed an even faster final ecdysis and adults are useful for mosquito taxonomical purpose.

Figure 1

The complete morphogenetic cycle of Aedes aegypti grown in fortified milk from daily sampling from zero to 240 h at 25°C and light-dark cycles of 12 h.

Figure 2

Solid yeast extract as an attractant for instar IV larvae of Aedes aegypti.

Figure 3

Quantitation of all mosquito forms at the seventh day of the Ae. aegypti morphogenetic cycle as a function of the nutrient sources (# = instar II; a = instar III; b = instar IV; c = pupa; d = adult; W = water; G = glycerol; Y = yeast extract; GY = glycerol + yeast extract; CNW = coconut water; LZM = lactose-free milk; SM = soy milk; WM = whole milk; LM = reduced-fat milk).

Figure 4

An adult Aedes aegypti on delivery from its mature pupa completing the mosquito morphological and physiological cycle.

Figure 5

Mortality percentages ± SE of Aedes aegypti instar IV larvae reared in coconut water killed by crude ethanolic extract of Piper nigrum fruit.