Collecting and processing specimens
Manual removal of calliphorid and sarcophagid larvae in cases of traumatic myiasis may be achieved by forceps. For calliphorid, sarcophagid and oestrid larvae in furuncles or warbles (e.g. Dermatobia, Hypoderma, Cordylobia, some Wohlfahrtia) extraction can be made by gentle pressure around the site of infestation, by occlusion of the opening (to prevent larval respiration), or by injection of anaesthetic (e.g. lidocaine hydrochloride) into the cavity below the larva to force it out. Note that insecticidal treatments after removal of larvae from wounds may have to be repeated at frequent intervals while the wound is healing to prevent reinfestations by calliphorids or sarcophagids.
Maintenance of living larvae
Adult flies are usually more easy to identify than immature stages, especially the younger immature stages. Therefore it can be valuable to keep some specimens alive, to rear to adulthood for a confirmatory identification. These should be placed in a jar with sawdust or paper tissue and a perforated lid (to enable gaseous exchange) and be maintained at room temperature. If the larvae are very young and of a facultative myiasis causing species, then some meat (e.g. liver, minced meat or canned dog food) should be added to enable them to feed and complete their development. Young larvae of obligate species are extremely difficult to rear to maturity. Adult flies can be killed by placing them in a deep freezer for one hour. With material reared from immature stages in the laboratory for identification, allow the adults to develop full colour and rigidity before killing them. Care should be taken not to keep dead, wet insects out of preservative at room temperature, especially in an airtight container, as they will quickly decompose if they cannot dry out. However, dry insects will keep well without preservative.
Killing and preservation of larvae
Direct immersion of live larvae into ethanol and other killing fluids causes larvae to contract as they die, making it difficult to see some diagnostic features. However, good quality, extended specimens can be obtained if larvae are first killed by immersion in near boiling water (>80oC) for 15-30 seconds (Adams & Hall 2003). Boiling water can be taken to the site of collection in a thermos flask or it can be prepared on site using a water heater operated from a car cigarette lighter. After killing, larvae should be transferred to 80% ethanol for long term storage. The preservative medium should be changed after 24 hours because it will be diluted somewhat by the water on and in the larvae.
Preparation of material for microscopical examination
The mouthparts, spiracles and other material should first be 'cleared' by macerating the specimen in a 10% aqueous solution of potassium hydroxide (KOH) at room temperature for at least 15 minutes. Specimens that have been in alcohol for six months or more need a longer period in KOH, up to 12 hours at room temperature or a shorter time in warmed KOH. Small larvae should be put into the solution whole, with punctures to allow its penetration; larger larvae can be dissected and only the required parts macerated, taking care to keep all the parts of a single specimen together. As the muscles soften they can be teased away with fine forceps or sharp needles. In order to avoid destroying the sclerites, care must be taken with dissecting instruments and with KOH. When the muscle and fat-body has been cleared away, the specimens should be placed in glacial acetic acid for at least 15 minutes, to neutralise any residual KOH, and should then be rinsed well with 80% ethanol. Thorough rinsing with ethanol should be sufficient if acetic acid is not available. They are then ready for examination, mounting or storage. There are many methods of mounting slides. One is to dehydrate the samples in absolute alcohol, transfer them to clove oil to clear, then mount in Canada balsam; alternatively, from absolute alcohol they can be mounted directly into Euparal.
Scanning electron microscopy
This can be a tremendous asset to the morphological study of fly larvae for identification and research purposes, the detailed images surpassing any produced by light microscopy (Spradbery, 1991). Careful preparation of the material is important to minimise physical distortion of the characters. Essentially, the material needs dehydration and then critical point drying before coating with gold/palladium.
Adams. Z.J.O. & Hall, M.J.R. (2003). Methods used for killing and preservation of blowfly larvae, and their effect on post-mortem larval length. Forensic Science International 138: 50-61.
Hall, M.J.R. & Vargas, M.T. (1990) In Manual for the control of the screwworm fly, Cochliomyia hominivorax, (Coquerel), B. Hursey (Ed.), Food and Agriculture Organization of the United Nations, Rome, Italy, 95 pp. Hall, M.J.R. & Smith, K.G.V. (1993).
Diptera causing myiasis in man. In: Medical Insects and Arachnids, Lane R.P. & Crosskey R.W., eds. Chapman & Hall, London, UK, 429-469.
Hall, M.J.R., Edge, W., Testa, J., Adams, Z.J.O. & Ready P.D. (2001). Old World screwworm fly, Chrysomya bezziana, occurs as two geographical races. Medical and Veterinary Entomology, 15, 393-402.
Spradbery, J.P. (1991). A Manual for the diagnosis of Screw-Worm Fly. Commonwealth Scientific and Industrial Research Organization (CSIRO) Division of Entomology, Canberra, Australia. 64pp.
Zumpt, F. (1965). Myiasis in Man and Animals in the Old World. Butterworths, London, UK, 267 pp.