Methods in Anopheles Research - MR4

Chapter 7 : Taxonomy and Systematics7.1 Methods for Collecting and Preserving MosquitoesPage 4 of 12adults for processing. To remove the viable emerged adult loosen the lid and carefully slip in and replaceit by the mouth of an inverted plastic holding vial. Tap on the side of the pupal container to induce themosquito to enter the holding vial and quickly stopper it with the original lid. Label this holding vial with thesame number that appears on the rearing vial containing the pupal skin. Remove the pupal skin with anapplicator stick (without dragging it along the side of the vial) and place it in the vial of 80% ethanol withthe associated larval skin. Completely fill the vial with ethanol to exclude air and attach it to the plasticholding vial containing the adult mosquito. This vial will now be processed as indicated below underREARED ADULTS. The entire process is repeated for all the pupal vials.All vials containing dead pupae and drowned, partially emerged adults stuck to the water should bediscarded. These specimens are not useful for taxonomic, but if they are alive they may be preserved forenzyme or DNA analysis.HOLDING VIALS. Reared adults for taxonomic study should be kept for at least 24 hours before beingkilled and processed as indicated in the section on REARED ADULTS. Many adults may die before theyare processed, in which case they will more difficult to mount on points on pins for study.The individual holding vials require relatively little attention. Check at least twice a day and process deadspecimens immediately or as soon as possible, following the directions in the section on REAREDADULTS. After the required 24-hour holding period, process the live specimens following the samedirections.PROGENY REARINGSGravid females collected in the field will generally oviposit within 3 to 4 days if they will lay eggs at all.Females should be retained in a cool damp environment and provided with 5-10% sucrose solution insaturated wads of cotton. The cotton wads should be moistened or replaced once or twice daily. On thethird day following capture, each female is lightly anaesthetised with ethyl acetate or knocked down in afreezer (about 1 minute) for the purposes of removing a wing. This is accomplished by grasping eachwing with a forceps and gently pulling until one of the wings is torn from the thorax. The anaesthetisedfemale is then placed on the surface of water (containing a hatching stimulus) in a small cup. Femalesstressed in this manner usually oviposit soon after being placed on the water. Some gravid females willrefuse to lay eggs and these can be preserved for enzyme or DNA studies. Once larvae become late firstor early second instars, they should be transferred to a larger bowl or pan and fed at least once per dayby sprinkling powdered food onto the surface. A portion of fourth-instar larvae (pre-pupae) should beremoved and reared individually for taxonomic study as described in the section on INDIVIDUALREARINGS. Remaining larvae can be preserved for enzyme or DNA analysis.KILLING AND PRESERVATIONEQUIPMENT AND SUPPLIES. The following equipment and supplies are needed for killing andpreserving mosquitoes: (1) killing tubes (preferably with ethyl acetate), (2) aspirators, (3) forceps, (5)labels, (6) 80% and 95% ethanol, (7) applicator sticks or scoop, (8) glass vials with lids, (9) beaker or panfor hot water.FIELD-COLLECTED ADULTS. Adult mosquitoes collected in the field are usually killed, identified andpreserved immediately on return to the laboratory. The method of preservation will depend on the type ofstudy they will be used for. Some blood-fed adults will be retained and set-up to obtain progeny broods(see PROGENY REARINGS).REARED ADULTS. All reared adults after being held for 24 hours in plastic vials (see HOLDING VIALS)should be killed and processed as indicated below. Parent females in PROGENY REARINGS should beprocessed immediately after oviposition following the procedure required for the type of study to beundertaken (i.e. morphology, enzymes or DNA).