machote




Protocols: The following are the main protocols selected by the Diptera TWIG for the present project. Protocol for Setting Up Malaise Traps and Handling Samples Protocols for Culicidae (Darlene Judd) Protocols for Ceratopogonidae (Art Borkent) Protocols for Dolichopodidae (Daniel Bickel) Protocols for Asilidae (Eric Fisher) Protocols for Sarcophagidae (Thomas Pape) Protocol for Acalyptratae (Stephen Marshall and Matthias Buck) Protocols for Ceratopogonidae (Art Borkent) Ceratopogonids, corethrellids and some chironomids (Stenochironomus complex) are placed in vials, sorted by the taxonomist, and then some returned for slide mounting and some for CPDing. Procedure for Slide Mounting of Ceratopogonidae The following procedure is similar to that for adults, with only a few modifications. It works well for specimens stored in ethyl alcohol and which are not too old (2-3 years or, if stored under dark and cold conditions, up to 7-10 years). To initiate the preparation for slide mounting put the following solutions into a series of stender dishes: 15% acetic acid, 2-propanol, 2-propanol layered over clove oil, clove oil (each dish about 3/4 full). If specimens are exuviae, the 15% acetic acid is not necessary. Each specimen should go through all above chemical solutions. All stender dishes should be placed in line on a piece of wood so that the can be moved together, and leaved all in a sheltered place while specimens are soaked. It is necessary to have a series of slides ready, which were cleaned with paper towel or with cloth. It is indispensable identify each slide with a label, and place them in a tray. For the first five slides the locality and specimen number are written so that they correspond to the first five specimens in preparation. If specimens are whole larvae or pupae (i.e. not exuviae) begin with step 1; if they are exuviae, begin with step 4. 1. Put a specimen into a dish of ethyl alcohol under the microscope. If the specimen is a whole larva or pupa, puncture the body wall with a very fine needle in several places in the thorax and abdomen (to allow KOH to more easily penetrate the body). One to many specimens from one sample are then placed in a vial of 8% KOH. 2. Place vials of KOH into a beaker partially filled with water. Place beaker on hot plate and heat water to boiling point. Specimens will clear (tissues will disintegrate) after 2-5 minutes, depending on the size of the specimens. It is important that specimens of a similar size are "cooked" at the same time so that they all clear at the same rate. 3. The following step requires some speed because specimens are damaged by being in hot KOH for too long. This is what needs to be done: remove the specimen from the vial into a dish with some 8% KOH in it and put under the microscope to see if it is properly cleared. The muscles should have dissolved and freely flow out of the specimen if squeezed very, very gently with the forceps (use this method only until you become familiar with what to look for; I never do this anymore). Also, if the specimens are over-cleared they will make for a poor microscope preparation. If cleared sufficiently, place specimen(s) in acetic acid. Leave for 15 minutes (or longer if necessary). 4. Move specimen(s) to 2-propanol for 15 minutes (or longer if necessary). 5. Move specimen(s) to 2-propanol layered over clove oil. Leave till specimen(s) has sunk to the bottom of the dish, which generally takes about 20-60 minutes, depending on the size of the specimen(s) (it can be left longer if necessary). After a day or two, the 2-propanol will mix with the clove oil and some more 2-propanol should be added so that there is always a distinct layer between the 2-propanol and the clove oil. If the clove oil has too much 2-propanol mixed into it, leave the dish without a lid for a few hours and the 2-propanol will evaporate from the mix. 6. Move specimen(s) to pure clove oil and leave for at least 30 minutes (or longer if necessary). 7. Place small drop of Canada Balsam on the microscope slide. Several larvae or pupae may be mounted on one slide, especially if one thinks that they belong to one species, but each specimen should be under a separate coverslip. Larvae should be mounted with the dorsum of the head directed upward. If the thorax and abdomen is not straight, it should be remove from the head capsule so that the apex of the abdomen is also positioned in a dorsal-ventral position. Pupae should have at least one respiratory organ removed (two if this can be done without damaging the cuticle from which the second respiratory organ arises). The pupal operculum should also be removed. The respiratory organ(s) and operculum should be placed under a separate coverslip (or a piece of a coverslip) and in such a position that, in case there are two or more specimens on one slide, that it is clear which dissected parts belong to which pupa.. The body of the pupa should be placed in a dorsoventral position with the dorsum directed upward and left to dry without a coverslip (so that the body will not be crushed). Once the Canada Balsam is at least partially hardened, a coverslip should be put on the pupa along with some extra Canada Balsam. In the upper right hand corner of the label put one or more of the following symbols: -_, _, P, L (for male, female, pupa, larva), depending on what is on the slide. 8. Put the slide into the drying oven. While the slide is drying, some of the Canada Balsam may leave a small air bubble under the coverslip. If so, add a tiny bit of Canada Balsam. Don't worry if the added Canada Balsam traps a bubble under the coverslip; the bubble nearly always moves out by itself (after some time). 9. Put the slide back into the drying oven. Check for air bubbles for the next couple of days and add Canada Balsam if needed (again, don't worry about trapped bubbles). Remove the slide after 2 weeks or so and put into a slide box. Keep the slide box upright, so the slides are still horizontal. Diptera TWIG / Sampling site selection / Taxa selection / Products / Protocols