Methods in Anopheles Research - MR4

Chapter 6 : Dissection Techniques6.6 Examination of Ovaries by Tracheal Distension to Determine ParityPage 1 of 46.6 Examination of Ovaries by Tracheal Distension to DetermineParityFrancis AtieliIntroductionParity is used to determine the age structure of a feral population, and it can also be used to ascertain thenet reproductivity of a colony (Githeko et al. 1993). Parous mosquitoes are those that have taken a bloodmeal and oviposited at least once. Nulliparous mosquitoes have never oviposited. Net reproduction rateof a colony can be determined by dissecting several females to determine the number of parousindividuals within the population.A more advanced technique was developed by Detinova (1962) that assesses how many egg batcheshave been developed by an individual female (reviewed by Hoc and Wilkes 1995). In this method, asembryos develop within the ovaries they stretch the ovariole sheath. After oviposition, these sacs whichcontain remnants from oogenesis shrink and develop into permanent dilatations. After each subsequentfeeding, a new embryo will form anterior to the previous dilatation. A mosquito with three dilatations wouldbe labeled “3 parous”. Caution should be taken when examining for dilatations as resorbed embryos willalso form dilatations. Likewise in some species such as An. atroparvus, dilatations may not be formed atall (Service 1993).The simplest technique for determining parity is to examine the tracheoles within the ovaries (Kardos andBellamy 1961). As the ovaries expand after the primary blood meal, the tracheoles that are associatedwith them are permanently distended (Hoc and Charlwood 1990). Therefore, in nulliparous females thetracheoles are tightly wound coils called ‘skeins.’ Parous females will have tracheoles that havedistended. Note that in younger females there may be a mixture of both skeins and ‘extended’ tracheoles;if any distended tracheoles are present, you can assume that the female is parous and has fed and laideggs at least one time.Materials• Microscope slides• PBS or another physiological dissecting solution• Forceps• Dissecting needles• Stereoscope• Compound Microscope (200 X magnification)Technique1. Gently anesthetize adult females.2. Place a drop of PBS on a clean microscope slide.3. Under the stereoscope, gently grasp female by the thorax with forceps, and place ventral side up withher abdomen in the PBS.4. While viewing the specimen under the stereoscope, take a fine tip needle or forceps and gentlyremove the 7th and 8th abdominal segments of the female by grasping them and pulling away slowly.5. Locate the ovaries; they will appear as a pair of white oval objects attached to the removed segments(Figure 6.6.1). Dissect away the accessory tissues and isolate the ovaries.6. Transfer ovaries to a new slide and allow to air dry.