MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 94(6) Nov/Dez 1999
PAGES: 845-847 DOI: Technical notes
A New Larval Diet for Colonization of Phlebotominae Sand Flies

Nataly A Souza, Claudia A Andrade-Coelho, Maurício L Vilela, André F Barbosa, Elizabeth F Rangel

Departamento de Entomologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil

Phlebotominae sand fly colonies are very important for studies of vector biology and the interaction of sand flies andLeishmania. Although, it requires excessive care, especially for immature stages, some sand flies species have been colonized in large numbers [R Killick-Kendrick et al. 1991 Parassitologia 33 (Suppl.1): 321-333].The larval diet is an important part of successful laboratory breeding due to the often excessive larval mortality caused by improper food.

MP Barretto (1942 Thesis Faculdade de Medi-cina USP, 162 pp.) suggested that during the immature stages period, the feeding preferences of sand flies from the New World may vary according to geographical location. However, the larval stage can use any kind of food, in situations where their prefered food sources are not available.

IA Sherlock and VA Sherlock (1959 Rev Brasil Biol 19: 229-250) made some experiments with Lutzomyia longipalpis in order to determine the most adequated culture conditions and medium for feeding immature stages. In these experiments decomposed organic vegetable matter products or dry bovine feces were the most suitable for larval development.

T Gemetchu (1971 Trans R Soc Trop Med Hyg 65: 682-684) tested liver powder as an alimentary diet of Phlebotomus longipes larvae. He observed that this feeding regime was better than a mixture of rabbit feces, and suggested that the duration of the larval period varied according to the composition of the diet. The liver power as larval diet was used by K Killick-Kendrick et al. (1977 J Med Ent 13: 429-440) and R Ward (Med Entomol 14: 469-476) too, for breeding of New World sand flies.

L. cruciata, L. anthophora, L. shannoni and L. vexator were reared in laboratory using the mixture of rabbit feces and laboratory rabbit chow as a feeding regime for larvae stages development. It was observed that larval mortality due to excessive fungal growth was reduced by ageing (or allowing the fermentation of the food) (DG Young etal. 1981 J Med Entomol 18: 446). After that, this larval food has been used in many laboratory colonies successfully (RG Endris et al. 1982 Mosquitos News 42: 400-407, R Killick-Kendrick & M Killick-Kendrick 1987 Ann Parasitol Hum Comp 6: 354-356,Killick-Kendrick et al. loc. cit.). B Modi and R Tesh (J Med Entomol 20: 568-569) used a modification of this mentioned larval diet. They added beef liver powder to the mixture of dried rabbit feces and rabbit chow.

In order to improve and optimize conditions for sand flies developing in laboratory colonies, we tested a new appropriate diet for immature stages. The sand flies were taken from our L. longipalpis laboratory colony, maintained according to ED Wermelinger et al. (1987 Mem Inst Oswaldo Cruz 82: 441-442), with the following modifications: after a blood meal, batches of 40 females were placed in rearing pots for oviposition and they were fed on a saturated sucrose solution according to NA Souza et al. (1995 Mem Inst Oswaldo Cruz 90: 751-757). The pots were observed daily and dead females were removed. The larval food was offered between 8 and 10 days after the transportation of engorged females to rearing pots.

Test diet - The larval food was prepared using a mixture of liver powder (Integalmédica, BZ Ind.), amino acids (histidine, isoleucine, cystine, methionine, tyrosine, cystine, leucin, glycine, proline, alanine, lysine, serine, valine - Probiótica, BZ Ind.), fish food, (Vitormônio, BZ Ind, E Rangel et al. 1985 Mem Inst Oswaldo Cruz 80: 219-226) and pollen grains (proteins, glicids, lipids, potassium, phosphorous, calcium, iron, vitamin B1, B2, C and B-carotene - Herbarium, BZ Ind.). Five grams of each component were used. After grinding, the components were mixed, sifted and distributed into small-sterilized glass tubes. For the third and fourth larval instars, the food was ready to be consumed. However, in the case of the first and the second instars, the larval food was sifted through a piece of nylon, before use so that we could obtain a very thin powder, since young larva have limited activity and they move slowly in the culture medium. In these experiments of 1,000 eggs each were treated by offering the test larval diet. In control batches fish food powder (Vitormonio) was used as diet, applied according to our breeding methodology (EF Rangel et al. 1986 Mem Inst Oswaldo Cruz 81: 431-438). The new larval food was tested for five consecutive life cycles with three repetitions each.

The obtained results revealed this food to be a satisfactory larval diet. Moreover, when compared with fish food the level of fungal contamination was greatly reduced, larval mortality decreased and output increased in relation to the number of adults emerged/number of eggs observed during five life cycles: 50.9, 38.3, 53.7, 52.3 and 48.8%. When fish food was used, the percentages of adults emerged were 24.9, 28.4, 20.4, 27.3 and 27.6% (Fig. 1). Significant differences were observed in terms of the duration of life cycle from eggs to adults, when the two larval diet were compared: (1) fish food: mean - 36.5, std. dev. 0.92 and (2) test larval diet: mean - 34.3, std. dev. 0.90 (Fig. 2). The good results obtained using this larval diet for L. longipalpis colony were observed with other Neotropical sand flies, such as L. intermedia and L. whitmani.

Souza et al. (loc. cit.) suggested that mix of sugars added to a source of amino acids could be the ideal diet for adult sand flies. We are of the opinion that its use combined with the new diet reported in this study and a source of amino acids could result in improved maintenance of sand fly colonies in the laboratory.

Acknowledgments: to Dr Gabriel Grimaldi Jr, Instituto Oswaldo Cruz, for contribution in the revision of the text and Prof. João Carlos Peixoto, Federal University of Rio de Janeiro, for the assistance with graphical illustration.

Work supported by Fiocruz and CNPq.
Received 6 October 1998
Accepted 12 July 1999

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