Methods in Anopheles Research - MR4

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Chapter 6 : Dissection Techniques6.7 Dissecting Spermathecae to Determine Insemination StatusPage 1 of 26.7 Dissecting Spermathecae to Determine Insemination StatusMR4 StaffIntroductionIn order to monitor colony mating activity and to assess wild-mosquito mating status, it is often necessaryto determine whether females are inseminated. The least complicated method to determine this is todissect spermathecae and determine whether spermatozoa are present. The spermatheca is located nearthe terminalia inside segment VIII and can often be seen through the cuticle while viewing the ventralside. It is a spherical, fenestrated dark-brown organ which has a golf-ball-like appearance. After gentlyremoving the terminalia and segment IX, the spermathecae will be exposed along with the malpighiantubules, ovaries, and digestive tract. Then the spermathecae can be easily removed and viewed undermagnification, and insemination status is usually readily apparent.Materials:• Fine forceps• Dissecting needles or minutien pin mounted on a long wooden stick• Clean microscope slides and cover slips• PBS solution• DropperEquipment:• Stereoscope• Compound microscope with dark field capacityProcedure:1. Anesthetize or kill the adult females.2. Place a drop of PBS on a clean slide.3. Gently grasp the female by the thorax with a pair of forceps and place ventral side up with theabdomen resting in the PBS under the stereoscope.4. Gently remove the terminalia of the female by grasping them and pulling away slowly using a fine tipneedle or forceps.5. Locate the spermatheca within the 8th segment and terminalia section removed previously. It shouldappear as a dark sphere that may or may not be surrounded by accessory tissues (Figure 6.7.1).Dispose of the mosquito carcass and remaining tissues.6. Gently lower a cover slip onto the spermatheca using a needle (to avoid rupturing the spermathecae).On the underside of the slide, circle the area surrounding the spermathecae using a permanentmarker. The marking will make it much simpler to locate the tiny organ. With experience, you willdetermine how much PBS to use to prevent rupturing the spermatheca.7. Under 100X magnification on a compound microscope, look for movement of the long thread-likespermatozoa within the spermathecae (Figure 6.7.2). They will appear as fine concentric threadswithin the spermathecae and are often seen rotating as a cluster. If the spermatheca ruptured afterplacing the cover slip, scan the surrounding field for the spermatozoa. Uninseminated females willhave a fairly transparent spermatheca (Figure 6.7.3).
Chapter 6 : Dissection Techniques6.7 Dissecting Spermathecae to Determine Insemination StatusPage 2 of 2Figure 6.7.1. Golf ball shapedspermatheca.Figure 6.7.2. 100X magnificationof a full spermatheca. The brightring is the bundle of sperm.Figure 6.7.3. Uninseminatedfemale spermatheca.
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\Methods inAnopheles ResearchSecond
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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Chapter 9 : Obtaining Live Material
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Chapter 9 : Obtaining Live Material
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Methods in Anopheles Research - MR4

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