Anopheles Pupa Collection and Sex Identification.

For most Anopheles species, larval-pupal metamorphosis commences ∼1 wk after egg hatching. However, depending on the amount of food provided, H2O temperature, and larval density, the pupation process can be accelerated or delayed. Synchronous pupation is difficult to accomplish consistently, and, thus, pupae need to be separated from larvae daily. Adult emergence will take place 24-48 h after pupation. Most adults will eclose before the next morning (light cycle) in many species. Here, we provide information on some methods available to collect pupae and to sort pupae by sex. Notably, pupa collection and sorting are some of the most time-consuming procedures of the overall mosquito rearing process. Some methods mentioned here attempt to help reduce work effort and time required.

Anopheles Egg Collection, Disinfection, and Hatching.

Gravid (i.e., with fully developed eggs), mated Anopheles females typically lay their eggs directly on water ∼48-72 h after a blood meal. Unlike some other mosquito species, Anopheles eggs cannot be desiccated and stored for long durations, and, hence, colonies must be reared continuously. In this protocol, we discuss methods for egg collection, including individual and en masse oviposition; egg disinfection to avoid the transmission of infectious agents to the next generation; and egg hatching for colony maintenance or experimentation. We also include optional methods for estimating life history traits such as fecundity, fertility, and larval mortality rates from egg counts.

Considerations for Rearing and Maintaining Anopheles in the Laboratory.

Anopheles mosquitoes can transmit several human pathogens, including viruses such as o'nyong-nyong and parasites including Plasmodium spp. and Wuchereria spp., which cause malaria and filariasis, respectively. Rearing Anopheles species of medical importance under laboratory conditions allows researchers to carry out experiments to better understand their genetics, physiology, and behavior. However, Anopheles species vary in how easily they can be reared in the laboratory, and some species have been difficult to colonize. Once established, members of the important African Anopheles gambiae complex thrive following a standard protocol and are predictable in growth and development rates. Here, we provide useful basic information and guidance to successfully maintain colonies of A. gambiae and other species of Anopheles in a laboratory setting. We also provide an example of a 3-wk rearing schedule that produces sufficient numbers of mosquitoes while minimizing the work required during weekends. In the accompanying protocols, we detail efficient methods and techniques suitable for several species of this genus at the egg, larva, pupae, and adult stages; however, it will be necessary for researchers to adjust methods as needed based on site-specific rearing observations of their particular strains.

Anopheles Adult Anesthesia, Feeding, and Sex Separation.

The adult stage is the only nonaquatic stage of the Anopheles mosquito. Both male and female Anopheles mosquitoes require access to a source of sugar to survive. In the insectary, a temperature of ∼27°C and 80% relative humidity and a cycle of 12 h light:12 h dark light, ideally with a sunrise and sunset period, are necessary minimum conditions to mimic their natural environment. Laboratory-reared Anopheles can survive for over a month; however, decreased activity and increased mortality may be observed ∼2 wk postemergence depending on the species and health of the colony. Details on how to maintain adults Anopheles are discussed here. Information and considerations on blood and sugar feeding are described. This protocol also provides instructions on how to differentiate male and female adult mosquitoes.

Anopheles Larval Rearing.

Mosquito larvae are aquatic and go through four development stages (larval instars L1-L4) before pupation. Species vary in the duration of larval development, and a variety of external factors affect the development rate (e.g., water temperature, food type, and larval density), which are discussed more thoroughly elsewhere. Here, we detail how to rear Anopheles larvae. This protocol describes appropriate distribution of larvae into rearing pans, feeding of larvae, cleaning of pans, and care until pupation.

Determination of Insecticidal Effect (LC50 and LC90) of Organic Fatty Acids Mixture (C8910+Silicone) Against Aedes aegypti and Aedes albopictus (Diptera: Culicidae).

Emerging and re-emerging vector-borne diseases such as chikungunya and dengue and associated Aedes vectors are expanding their historical ranges; thus, there is a need for the development of novel insecticides for use in vector control programs. The mosquito toxicity of a novel insecticide and repellent consisting of medium-chain carbon fatty acids (C8910) was examined. Determination of LC50 and LC90 was made against colony-reared Aedes aegypti (L.) and Aedes albopictus (Skuse) using probit analysis on mortality data generated by Centers for Disease Control and Prevention bottle bioassays. Six different concentrations of C8910 + silicone oil yielded an LC50 of 160.3 µg a.i/bottle (147.6-182.7) and LC90 of 282.8 (233.2-394.2) in Ae. aegypti; five concentrations yielded an LC50 of 125.4 (116.1-137.6) and LC90 of 192.5 (165.0-278.9) in Ae. albopictus. Further development of C8910 and similar compounds could provide vector control specialists novel insecticides for controlling insect disease vectors.