Methods in Anopheles Research - MR4

Chapter 7 : Taxonomy and Systematics7.1 Methods for Collecting and Preserving MosquitoesPage 3 of 12and lids, (8) 80% and 95% ethanol, (9) labels and (10) pencils, grease pencils. All the equipment shouldbe for use exclusively in the laboratory and should not be taken into the field.CARE OF COLLECTIONS AND SORTING. Immediately after a field trip all the collections must bechecked carefully. Labels on all containers should be made plainly visible and legible.Set up immature collections by emptying contents of collection bags and vials into separate bowls. Labeleach bowl with the same number marked on the collection bag or vial. The sorting of the immature stagesand their separation for individual rearings and preservation can now be carried out. The sooner this isdone the better, and this should always be completed within 24 hours of capture. Work with onecollection at a time going through the whole process before turning to the next collection. If more thanone collection bag or vial contains immature stages from the same collection, assemble them all together.Generally, more than one species is found in a single collection of immature stages even whencollections are made from individual breeding sites, but as a rule one species is dominant in a particularcollection and the others are frequently represented by few specimens or by younger larval instars.First, all the pupae are transferred one to a plastic vial in about 2 cm (somewhat less than one inch) offresh clean water (always use rainwater or distilled water). The plastic vials are marked with thecollection number (grease or wax pencil is preferred but paper labels can be used). These will be laterprocessed as indicated below under EMERGENCE VIALS. As a rule, all the pupae present in a collectionshould be isolated individually unless the collection consists primarily of pupae and the total numberexceeds 10. If it is obvious that several species are represented among the pupae, a larger numbershould be isolated individually (see EMERGENCE VIALS below).Second, the fourth-instar larvae are picked up one by one and placed into separate plastic vials. Isolateall mature larvae in the collection, or a maximum of 10 per species if the collection is very large. Theindividual vials are filled with distilled water or rainwater to a height of about 2 cm (somewhat less thanone inch), marked with the collection number in grease pencil and set aside to be processed as indicatedbelow under PUPATION VIALS. The remaining larvae in each collection, normally up to a maximum of20, are set aside for killing and preservation (see WHOLE LARVAE in the section on KILLING ANDPRESERVATION), and any larvae left over are retained in the bowl and removed for individual rearing asthey become fourth instars. In case it is not practical or desirable to take care of a large numbers ofcollections or rearings, all the larvae remaining after isolation of individuals should be killed and preserved(NEVER DISCARD ANY MATERIAL ONCE COLLECTED). Great care should be taken to thoroughlyrinse sorting pans and pipettes between the processing of different collections to eliminate contamination.WASHING CONTAINERS. All containers used for collecting, sorting and rearing must be washedthoroughly before they are used again. First, wipe off the grease pencil markings (if used) from the drybowls and vials with a piece of cotton or paper. Containers that are reasonably clean may be merelyrinsed several times in clean fresh water. Aspirators should also be cleaned periodically and the nettingreplaced on the plug.PUPATION VIALS. Vials containing isolated larvae, marked with collection numbers, should beexamined twice a day for pupation, preferably in early morning and late afternoon. Check through all thevials and set aside all those containing larval skins before further processing. Remove the larval skin withan applicator stick (without dragging it along the side of the rearing vial) and transfer it into a storage vialwith 80% alcohol and attach it with an elastic band to the rearing vial containing the pupa. Place a lid onthe rearing vial to prevent escape of the adult mosquito after it emerges from the pupa. This marked vialis now set aside to be processed as indicated under REARED ADULTS in the section on KILLING ANDPRESERVATION below.EMERGENCE VIALS. Vials containing isolated pupae should be examined twice a day, preferably inearly morning and late afternoon. Check all the vials and set aside all those with emerged or drowned