Sexing sand fly pupae (Diptera: Psychodidade: Phlebotominae)

Abstract

We describe a technique to separate male and female pupae of sand flies. This has reduced the labour to separate flies after emergence and also allows the isolation of unmated adults for behavioural and physiological studies.Key words: Insecta -sand flies -sex -pupae -morphology Manipulation of adult sand flies involving aspiration and transfer between recipients is stressful to the insect and the separation of large numbers of male and females is a laborious and timeconsuming job. Damage to the insects is also undesirable when these are used in morphological studies. Although separation of the sexes in the immature stages is very difficult for most insects, it is easily achieved for phlebotomine sand flies. Morphological differences in the last segment of male and female pupae of sand flies were pointed out by Barreto in 1940 and also by Mangabeira (1942a,b) when studying the life cycle of few Brazilian species. Since these observations were published, distinction of the sexes at the pupal stage has not been pursued by sand fly researchers, although advances have been made in the colonisation of various species during the last decade, making it possible to focus attention on different aspects of the biology of these insects. Researchers are now concerned with behaviour as well as with the morphophysiology of the male and female besides the role of the different species as vectors of the disease. Thus, a routine procedure for sexing sand fly pupae becomes a standard requirement to obtain adult insects for use in these studies (Ward et al. 1987(Ward et al. , 1990 The following materials are required for this work: a large petri dish lined with a disc of filter paper, a soft thin paint brush (no. 0), a small container of distilled water in which to dip the paint brush, and a stereo microscope.Procedures: (1) pupae are obtained by any of the methodologies already established for the colonisation of sand flies (Modi & Tesh 1982, Brazil et al. 1997; (2) the filter paper is moistened with distilled water; (3) pupae are transferred from the breeding pots with the help of the paint brush and left for 5 sec to 10 min to moisten the larval exuvia which remains attached to the last pupal segment; before collecting each specimen the paint brush must be dipped in distilled water; (4) the larval exuvia is gently removed with the help of the paint brush so that the last segment of the pupa is revealed. Manipulation of the pupae should be done under the stereoscopic microscope. The separation of male and female pupae involves examination of the last pupal segment which in the male is larger, more rounded and paler than the previous one (Figure). The female body segments decrease in size from the cefalothorax to the tip of the abdomen, so that the last abdominal segment is the smallest of all. This difference in segment proportion is due to the relatively long size of the male external genitalia, consisting of paired styles, coxites, lateral lobes and parameres. After

Efficiency of the separation of Aedes (Stegomyia) albopictus (Diptera: Culicidae) male and female pupae using a sieving device

“…Separation of unmated individuals by sex in the adult stage may be difficult because mating can pass unnoticed, forcing the researcher to be aware of the moment they molt into adults to be rapidly removed (Brazil & Brazil 2000). For small experiments this can be a manageable task, by placing larvae or pupae in separate vials (Dame et al 1974).…”

Preparation of sand fly (Diptera: Psychodidae: Phlebotominae) specimens for histological studies

“…When pupation occurred the specimens were sexed as described by Brazil and Brazil (2000) and transferred individually to emergence vials. The vials were numbered and emergence of the insects monitored at hourly intervals.…”