Methods in Anopheles Research - MR4

Chapter 7 : Taxonomy and Systematics7.1 Methods for Collecting and Preserving MosquitoesPage 6 of 12is far better to tape a label to the outside of the vial, or to write the specimen number on a piece of tapeand wrap it around the vial in a lightly overlapping manner to enhance adhesion.PRESERVATION AND MOUNTING TECHNIQUESWHOLE LARVAE1. Kill larvae in hot water (not boiling), remove promptly with a lifter. Store in small vial containing 80%ethanol (ethyl alcohol). For material to be used for DNA studies, larvae should be killed by placingdirectly into 95% ethanol – DO NOT kill in hot water.2. Transfer specimens from alcohol to cellosolve for 15 minutes or more (dark specimens can be storedin cellosolve for 8 hours or overnight).3. Lift the specimen from cellosolve and place on the centre of glass microscope slide with the dorsalside up.4. Drop a small amount of Euparal on the specimen. Mount specimen dorsal side up with the headpointing down; arrange head, thorax and abdomen in natural position, then cut the abdomen betweensegment VI and VII (with a fine scalpel blade). Place the terminal segments with siphon to the left inculicine larvae or segment X to the right in anopheline larvae (see FIGURE 7.1.3).5. Place more Euparal on the specimen and check the arrangement of setae and larval position, thencarefully cover the specimen with a 22 mm rectangular cover glass.6. Dry in an oven at 45º-55ºC for 4 weeks or more.LARVAL AND PUPAL EXUVIAEThe fourth-instar larval and pupal exuviae from an individually reared adult should be mounted on thesame slide.1. Store in 80% alcohol.2. Transfer the specimens into cellosolve for 15 minutes.3. Lift the specimen from cellosolve placing it on a glass microscope slide, the larval exuviae on the leftand pupal exuviae on the right (pointing head down and dorsal side up).4. Drop a small amount of Euparal on the specimens. Arrange and spread the body and setae of larvalexuviae into better position, then separate the pupal cephalothorax just cephalad of the wing, leavingthe metanotum attached to the abdomen. Open the cephalothorax, mount it ventral side up andplace it below the metanotum (see FIGURE 7.1.3).5. Add more Euparal, check the position of the exuviae then cover the specimens with a 15 mm roundcover glass.6. Dry in an oven at 45º-55ºC for 4 weeks or more.ADULTS1. After emergence adults should be held for at least 24 hours before killing.2. Kill in a killing tube charged with ethyl acetate or chloroform 1 (ethyl acetate is preferred because itkeeps specimens relaxed longer).3. Apply a small amount of Ambroid ® cement 2 or other glue to the tip of a paper point and affix thespecimen on the right side of the thorax with the legs toward the pin (see FIGURE 7.1.3).1 CAUTION! Chloroform and Ethyl Acetate are toxic and dangerous to breathe. These chemicals are stored in liver tissues andmay cause health problems if used frequently. Always use in well ventilated areas.2 Ambroid ® cement should be thinned down with amyl acetate.