The basic rules and methods of mosquito rearing (Aedes aegypti).

The rearing of Aedes mosquitoes is complex and demanding for several reasons. Aedes larvae are affected by temperature, density and available nutrition, mating is not necessarily accomplished naturally and females need a blood meal to develop eggs. The climate chambers where the mosquitoes are kept are warm and sweaty. Due to these tropical conditions the larvae develop fast and need to be cared for daily. The Laboratory of Entomology in National Institute of Malaria Research Bangalore has cultured different colonies of different vectors successfully. In this paper, we discuss different aspects off the rearing process which affect mosquito fitness and are of importance for the quality of fundamental and applied research.

INTRODUCTION

Aedes aegypti, a vector of dengue, is widely distributed in the tropical and subtropical zones. About two-thirds of the world's population lives in areas infested with dengue vectors, mainly A. aegypti. Dengue viruses infect over 100 million people every year.[1] Possibilities to reduce dengue incidence are studied world-wide. Vector control is one of the options, as interruption of transmission of dengue parasites is clearly the most effective disease control strategy. In laboratories, mosquito colonies are needed in order to conduct studies on vector biology, vector-parasite interactions, insecticide susceptibility, vaccine studies etc., For this reason it is important to maintain the original gene pool, physiological and behavioral characteristics of the insects under study as much as possible. A healthy insect colony does not only increase the possibilities for successful experiments in the field, it also reduces the chance of contradicting results. The quality of experimental insects can be influenced by population density, food availability, climatic conditions, etc., These factors can strongly influence the outcome of experiments and should therefore not be ignored.[23]

THE STUDY

The Aedes mosquito rearing at National Institute of Unani Medicine Bangalore takes place in culture rooms that simulate natural climatic conditions. Temperature is set at 27°C ± 2°C and relative humidity at 75% ± 5% respectively. Temperature and humidity controls are probably the most important factors in the successful rearing of mosquitoes. The simplest method of providing regulated heat and humidity is the use of a small electric light bulb and a wet towel draped over the cage. The most elaborate is a complex environmental chamber with programmed electronic controls of temperature, humidity and photoperiod. The size of the insectary will regulate the type of temperature and humidity control system required. The photoperiod and light intensity affect the development of the various stages in the life cycle of the mosquito and is the subject of a great many papers. In the insectary, a cycle of 14 h of light and 10 h of darkness appears to allow the best and most uniform development.[45]

Eggs are collected on moist filter paper. A small bowel is lined with a 3″ wide strip of filter paper. Water is added to a depth of 2.5 cm. The container is then placed in a cage of adults, the egg collecting container is left in the cage for 48 h. The bowel is removed and any excess water is drained out of the bowel. The egg paper is allowed to remain an additional 24 h in the cage, then removed and air dried for 4 days then stored by placing them in a large sealed plastic container. The eggs are hatched in deoxygenated. The water temperature should be 27°C (80°F). The larvae hatch in 6-12 h. approximately 70-95% hatch is expected on eggs that are < I2 months old. The larvae are counted by the aliquot method. Approximately 67 larva/cm2 of water surface with a depth of 1.5 cm should be introduced into the tray. A plastic tray (20 cm × 15 cm × 5 cm) is satisfactory to rear 1500 larvae. The time required to complete larval development varies from 7 to 23 days depending on temperature, food and density of larvae.

Water in rearing container was refreshed every day by removing a little quantity of water from the rearing tray and replacing with fresh water. This was aimed to prevent scum formation on the water surface. Pupae were collected from day 7 to 15 and placed in emergence cages where they emerged. When all the adults have emerged, the cover is replaced on the plastic container. Approximately 2000 pupae are placed in a cage 30 cm × 30 cm × 30 cm (12" ×12" ×12"). A ratio of 3 females to 1 male is preferable. This gives a density of 0.5 mosquito/cm2 of vertical resting surface or a density resting surface of 1.85 cm2 of resting surface per mosquito.[6]

Investigators have recommended various materials as larval diet. These may be classified into the following categories. Mainly carbohydrate or its products: Bread, powdered rice, wheat bran flour, maize, rye, soya bean powder; Animal protein: Dried serum, dried blood, powdered liver, shrimp powder, skim milk; Artificial diet: Squarium diet, fish food (Tetramin), dog biscuit, rabbit-feed pellet, mouse-feed pellet; Yeast: Baking yeast, powdered brewer's yeast. Infusion: Hay infusion, bread infusion, wheat infusion, guinea-pig pellet infusion; Others: Guinea-pig faeces, corixid powder, Chlorella vulgaris, etc., As regards the controlled feeding of A. aegypti. Larvae, Burchfield (1958) and his co-workers have proposed a very elaborate technique. For the purpose of delicate screening work such precise methods and the use of a strictly controlled laboratory strain may be required, but for the present only a method that can be performed in any laboratory in the world will be considered. First of all, the diet itself should be readily available, convenient to treat, easy to preserve and cheap.[7] Keeping this fact in mind we used “tetramin” fish food and yeast tablet.

Although the female mosquito usually must ingest a blood meal for ovarian development, adults of both sexes require carbohydrate foods in addition. Carbohydrates are generally supplied as a sugar solution. Although sucrose and glucose in concentrations of from 3% to 20% have been used, 10% sucrose, made by dissolving 100 g of ordinary household white sugar in 1 L of water appears to provide the best results. Other forms of sugar, such as corn syrup, honey, various fruit juices, raisins, apple slices and bananas, have also been used. Soaked cotton balls are the easiest method of providing the sugar solution to adult mosquitoes. The cotton balls are soaked in the 10% sugar solution, the moisture is squeezed out and the balls are then placed on the top of the cage. Usually 4 cotton balls, changed daily, are sufficient for a 30 cm × 30 cm × 30 cm (12 × 12 × 12") cage. The cotton balls must be changed daily. The more commonly used sources of blood meals, other than a technician's bared arm, are guinea pigs, rabbits, mice, rats, hamsters, monkeys and chicks. Blood meals may also be supplied by artificial means. Shave the animal prior to presentation to the mosquitoes. Some prefer shaving or clipping the hair from the back of animals and others the underside. Animal clippers' have proved very useful for this purpose.[8]

CONCLUSION

The acquisition of knowledge concerning proper control of mosquitoes and mosquito borne diseases requires that studies be made of the biology, physiology, anatomy, genetics, taxonomy and ecology of the insect. Insecticide resistance, biological, chemical and integrated control must be investigated. The use of mosquitoes as screening agents for pesticides or chemotherapeutic compounds requires large numbers of mosquitoes. All phases of mosquito research usually use individual or quantity rearing of mosquitoes. Mass rearing of mosquitoes for control purposes may become in the near future as common as mass production of pesticides.[9] This paper describes only a few examples of measures taken during the rearing process to guard the fitness of the mosquitoes. Colleagues working in different disciplines and with different organisms may, however, encounter comparable trade-offs during the design of their culture. Laboratory conditions are usually different from field conditions. For this reason, ecologists who conduct laboratory experiments with living organisms should be cautious in the application of their results to a field situation.

The purpose of this paper is to provide information on the rearing of mosquitoes. No method for rearing mosquitoes is guaranteed. Perhaps the most important requirement for successful rearing of mosquitoes is attention to detail. Mosquito rearing to be successful requires attention 24 h a day, 7 days a week. The basic rules are: Avoid over-crowding of mosquitoes and overfeeding larvae, avoid pesticide contamination, observe temperature and humidity requirements, standardize rearing methods and avoid non-standardized food. Observance of these principles should produce uniform animals.