GTF 511: Cephalopharyngeal acquisition Introduction In a forensic investigation, analysis of maggots is useful to determine the species of fly for determining the post-mortem interval. Aside from the overall morphology, spiracles and spine bands, the cephalopharyngeal skeleton could be used for species identification. It is located in the anterior segments of the larvae and consists of the mouth hooks which are employed for feeding and locomotion purposes. The cephalopharyngeal skeleton furthermore provides attachment sites for the pharyngeal muscles. The skeleton can be obtained by dissection. The cephalopharyngeal skeleton of the internal structure located at the anterior end of a maggot. Figure 1 shows the dorsolateral view of the whole larva. Figure 2 shows the lateral view of the cephalopharyngeal skeleton presenting the clipeal arc, dorsal cornu, concavity of pharyngeal sclerite, ventral cornu, base of parastomal bar, union between hypostomal sclerite, apical hook and dorsal apodeme of mouth hook. Figure 1: Dorsolateral view of the whole larvae
Materials and methods Table 1. List of analytical apparatus Table 2: List of chemicals and reagents No. Items Description No. Items Description 1. Stereomicroscope Model: SMZ-168 BH LED; Brand: Motic 2. Laboratory Waterbath Model: WB 29; Brand: Memmert 3. Vortex Mixer Model: S0200-230V; Brand: Labnet 4. Leica MZ16 stereomicroscope with Leica MC170 HD digital microscope camera Leica Microsystem, Heerbrugs, Switzerland Figure 2: Lateral view of cephalopharyngeal skeleton (Nuñez and Liria, 2016).
1. Sodium Carbonate (Na2CO3) Sigma-Adrich, Japan 2. Bleaching liquid (Clorox) Clorox Professional Products Co 3. 15% Sodium Hydroxide (NaOH) Merck, Germany 4. 70% Ethanol Teraslab Saintifik Sdn Bhd, Malaysia Table 3. List of materials used No. Items Description 1. Beakers 250 mL 2. Beakers 100 mL 3. Beakers 25 mL 4. Petri dishes (Plastic) Aided in placing maggots from preservation storage 5. SCHOTT bottles (500 mL) Storing maggots 6. Measuring Cylinder 100 mL 7. Forceps To remove maggots 8. Applicator Sticks Dissect the maggots 9. Tissues Absorb moisture from maggots 10. PYREX® disposable Culture Tubes To place the cephalopharyngeal skeleton for vortex 11. PYREX® disposable Culture Tubes holder To place the tube in waterbath 12. Superior latex exam gloves Precaution 13. Plastic dropper Preparing cleaning solution 14. Cellophane tape Aid in dissection 15. Adobe Photoshop software Adobe Photoshop CC 2017
Methods 1. Place a tissue paper on a petri dish which will absorb the maggot’s moisture in order to facilitate the dissecting process. 2. Take one dead maggot from the bottle containing 70% ethanol with a forceps/tweezers onto a petri dish. 3. Identify the morphological appearance of the maggot species using microscope. a) Sponge b) forceps c) Modified wooden applicator stickwith sharp pointer d) Needle
Obtaining the cephalopharyngeal skeleton The procedure to acquire the cephalopharyngeal skeleton are as follows:
1. Cellophane tape is utilised on the stage of stereomicroscope to facilitate the process of dissection of maggots. 2. The anterior segments of the maggots is dissected using a modified sharp blade aided using modified sharp needle together with a wooden applicator stick to acquire the cephalopharyngeal skeleton. 3. The acquired cephalopharyngeal skeletons is placed in test tubes containing distilled water. 4. The solution for cuticle softening that attached onto the cephalopharyngeal skeleton is prepared by using 3.75 gram of sodium carbonate (Sigma-Adrich, Japan) and 1 ml bleaching liquid (Clorox Professional Products Co) and then dissolved in 29 ml distilled water. Next, the combined chemicals are dissolved in 20 ml of 15% sodium hydroxide (Merck, Germany). The total volume of the mix solution is 50 ml. 5. The acquired skeletons are placed into the test tube containing the solution and are incubated at 80°C in a water bath for 10 minutes. 6. The solution containing cephalopharyngeal skeletons is then vortexed for 3 minutes. 7. The cephalopharyngeal skeletons are taken out from the test tube and washed using distilled water. 8. The Leica stereomicroscope is utilised to observe the image of the skeletons and photographs are acquired.