Methods in Anopheles Research - MR4

Chapter 6 : Dissection Techniques6.5 Dissecting Plasmodium-Infected Mosquitoes6.5.1 MidgutPage 1 of 26.5 Dissecting Plasmodium-Infected Mosquitoes6.5.1 MidgutMR4 StaffIntroductionExperimental Anopheles infections may be performed todetermine the length of sporogony for various Plasmodiumspecies or to establish susceptibility or refractoriness toPlasmodium infections. Dissection of the midgut to observeoocysts should be performed 5-7 days after infection occurs.The precise number of days that is best will vary with parasitespecies. Some examples of variability are shown in Table6.5.1.1 (WHO 1975). An alternate method by Looker andTaylor-Robinson can be found in Methods in Malaria Research(Looker and Taylor-Robinson 2004).SpeciesP. vivax 4-7P. ovale 3-4P. falciparum 3-7P. malariae 3-11Days to oocystsTable 6.5.1.1. Adapted from WHOManual on Practical Entomology, 1975.MaterialsStereoscopeDissecting needlesMicroscopeForcepsPBS or saline solutionMicroscope slidesMercurochromeCover slipsChloroformDetecting the presence of Plasmodium oocysts in the midgut:1. Anesthetize the females (see section on Mosquito Anesthesia).2. Place a drop of PBS on a clean microscope slide.3. Place a female in the drop of PBS ventral side up with the terminalia in the center of the drop.4. Grasp the female by the thorax using forceps.5. Lay a dissecting needle across the 7 th abdominal segment (without slicing the abdomen) and gentlydetach the terminalia.6. While still grasping the thorax with forceps, gently pull the terminalia away from the abdomen using thedissecting needle. Pull very slowly to ensure that the midgut does not detach from the terminal end(Figure 6.5.1.1).7. If the midgut detaches, gently cut the edges of the 1st abdominal segment and gently pull the cuticleover the contents.8. Remove the Malpighian tubes as well as any other accessory tissue and debris leaving only themidgut.