Methods in Anopheles Research - MR4

Chapter 7 : Taxonomy and Systematics7.1 Methods for Collecting and Preserving MosquitoesPage 5 of 12Process all the adults (dead or alive) from individuals that have been held for the length of time (24 hours)together at some convenient time in the afternoon or evening. Numbers are assigned to individualspecimens at this stage, and the necessary information is recorded on the appropriate collection andrearing form (see Figures 7.1.1 and 7.1.2). For example, an adult female with larval and pupal skins isgiven the next available number on the form, perhaps 12-2, indicating that this is the second specimenreared from collection 12. The first specimen from this collection would have been labelled 12-1. Thenumber needs to be written on 2 labels, one for the adult and one for the vial containing the associatedskins. Pre-printed labels may be used to avoid errors and increase efficiency.To speed the process, have at hand 2-5 killing tubes to be used serially. Loosen the lid of a holding vialand holding the vial upside down slip its mouth over that of a killing tube. The adult will be knocked downby the ethyl acetate within seconds and will fall into the killing tube. To hasten knockdown, tap on thevial. Remove the vial and hold a thumb over the mouth of the killing tube until the specimen ceasesstruggling. Place the label inside the killing tube, stopper the tube and set it aside. Proceed in the samemanner with 1-4 other adults and place the killing tubes in sequence. After all the tubes are used, returnto the first one and process it as indicated in the next paragraph. After this step is finished kill anotheradult with the emptied tube and place it in sequence after the others. Continue in this manner until all theadults are processed. Time the processing to allow the adult to remain in a tube about 5 minutes, but notlonger than 10 minutes by using the appropriate number of killing tubes.Transfer the adult and its label from the killing tube to a clean white card. Using fine forceps pick up theadult by one leg and place it on an elevated surface (e.g. empty slide box) with a white background.Orient the specimen with its left side facing down. This should be accomplished without grasping thespecimen with the forceps. Move the specimen to the edge of the surface with its legs projecting beyondthe edge. Insert a heavy paper point an appropriate distance from the head of an insect pin and place atiny droplet of ambroid ® cement or other suitable glue on the upper apical angle of the point. Holding thepin so that the point is upside down, gently touch the droplet of the glue to the thorax (right side) of themosquito. Final orientation of the mosquito on the upper surface of the point is with the left side up, headfacing left and the legs extended toward the pin. This orientation protects the specimen from damage andcorresponds to the preferred orientation of illustrations in taxonomic publications. Place the label with thecollection and rearing number on the pin and store the specimen in an appropriate insect storage box.WHOLE LARVAE. It is essential to preserve an adequate sample of whole larvae of every species fromevery field collection and from all progeny rearings (see CARE OF COLLECTIONS AND SORTING in thesection on INDIVIDUAL REARINGS). To be useful for taxonomic purposes, whole larvae must be killedand preserved carefully so that the body shape and all structures, particularly setae, are retained. Thelarvae set aside for killing and preservation (identified by the collection number) are first transferred with adropper to a small cup or bowl with fresh clean water as a washing procedure. If much debris or sedimentis still present, additional serial transfers should be made until it is eliminated. Remove as much water aspossible from the cup or bowl using a fine pipette. Next heat a beaker or pan of water is about 60ºC(140ºF) and pour the hot water into the cup or bowl. As soon as the larvae float up to the surface, thewater is removed with a fine pipette and replaced with a quantity of 80% ethanol. After 5 minutes thelarvae are transferred with a lifter (do not use forceps) to a vial with 80% ethanol. Completely fill the vialwith 80% ethanol to remove all air and cap it tightly. No more than 20 larvae should be placed into asingle vial as the water contained in the bodies of the larvae will significantly dilute the concentration of asmall quantity of ethanol and jeopardise preservation. Prepare a paper label in pencil and tape it to a vial.The label may be placed inside the vial if the larvae are separated from the label by a small wad of cotton.Larvae to be used for DNA studies should ONLY be killed by placing them into a vial of 95% ethanol.SKINS. The most valuable material for taxonomic purposes is the associated larval and pupal skins fromindividual rearings and the corresponding adults. The greatest care must be taken in processing these.Labels should not be enclosed in vials with skins unless these are separated by a small wad of cotton. It