Chronometrical Morphology Changes Of Larval Cephalopharyngeal Skeleton And Non-Specific Esterase In The Different Developing Stages Of Parasarcophaga Crassipalpi (Diptera: Sarcophagidae) And Their Implications In Forensic Medicine

Objective: To study the growth and development patterns of the larval cephalopharyngeal skeleton and the changes of non-specific esterase of Parasarcophaga crassipalpi under different temperatures and accumulate biological data on necrophagous flies in Shijiazhuang area. These data would help provide scientific evidence for deducing the postmortem interval (PMI) in criminal investigation.Methods1 Preparation and observation of experimental specimen Adult Parasarcophaga crassipalpi were collected from the outdoors. The experiment was done with offsprings of the third generation. They were reared successively in different biochemical culture boxes under the temperature of 16℃, 20℃, 24℃, 28℃and 32℃respectively. The humidity, photoperiod and food supply were kept unchanged during the entire study. When larvae were laid, 10 of them were randomly sampled from each group at 12h intervals until the beginning of the prepuparial stage. The larvae were fixed in boiling water and then preserved in 70% alcohol mixed with glycerine. The samples were taken out from the preserved solution and the liquid of surface was blotted up by the filter paper. The innards of specimen were corroded with 10% KOH and dehydrated with alcohol from 30% to 100% gradually. After being transparent with dimethylbenzene, the specimens were observed and photographed. However, other specimens which were used to measure up should be detached symmetrically under microbioscope. Finally the indexes including areas of different parts of cephalopharyngeal skeleton and average optical density had been given by digital image processing equipment.2 Detection of non-specific esterase activityCleaned by phosphate-buffered saline (PBS) three times, the larva was placed in Eppendorf tube under 20℃below zero. After homogenation and centrifugalization at low temperature, supernate fluid was extracted as esterase source for further use. 100μl fluid and the same amount ofβ-NA solution as the substrate were put into every hole of microtiter plate to react under 25℃for 10 minutes. Then 100μl fast blue B salt solution as the color agent was added to each hole. The microtiter plate was put under room temperature for 5 minutes. By means of using automated immuno analyser, the OD values were detected to reflect NSE activities during different stages of larva.Use analysis of variance (ANOVA) when made statistical analysis different indexes of cephalopharyngeal skeleton and the OD values. Finally the data were statistically curved with Excel.Results1 Under the same temperature condition, the colour of each part of cephalopharyngeal skeleton became deeper than ever during the growth of larvae. The degree and range of chitinazation in cephalopharyngeal skeleton increased along with the development.2 In the same temperature, the area of mouth hook increased periodically. The changes usually happened when the instars overlapped. Once got the maximum, it stopped increasing. The growth speed of the area of mouth hook was faster and the maximum appeared earlier at higher temperatures. The average optical density and the sclerotized area shared the same developing rate. However, there were more variations within the two indexs in the first and second instar in comparison with those of larval mouth hook. Sclerotic degree of mouth hook kept increasing within single instar. Once entered the next stage, the index began growing from a lower point. The growth of angle between ventral part and base part of larval mouth hook decreased in period as larva grew.3 In the same temperature, the area of larval pharyngeal sclerite also increased periodically. However, The average optical density and the sclerotized area of pharyngeal sclerite grew gradually all the time and almost at the speed to the end of third instar. Given higher temperatures, the speed of these indexes was faster and the maximal data arrived at a earier stage. Sclerotic degree of pharyngeal sclerite had the similar growth rate to that of mouth hook.4 In the same temperature, the OD value which reflected NSE activity was growing everyday during the first and second instar. At the beginning of third instar, it still kept increasing till the maximum arrived. After that, it began to drop slowly. Yet the OD value of the end of third instar was much higher than the maximum of the second instar.Conclusions1 Under the same temperature condition, there were regular changes in the larval growth and development patterns of the Parasarcophaga crassipalpi during different stages.2 Among the morphology indexes, the indexs of mouth hook kept growing periodically. Sclerotic degree of mouth hook kept increasing within single instar. Once entered the next stage, the index began growing form a lower point. The growth of angle between ventral part and base part of larval mouth hook decreased in period as larvae grew.3 The average optical density and the sclerotized area of pharyngeal sclerite grew gradually all the time and almost at the speed to the end of third instar. Changes in area and sclerotic degree of pharyngeal sclerite were in similar way as those of mouth hook.4 The OD value which reflected NSE activity was growing incontinuously. At the beginning of third instar, it still kept increasing till the maximum arrived. After that, it began to drop slowly. Yet the OD value of the end of third instar was much higher than the maximum of the second instar.