Pupal Morphogenesis Of Lucilia Cuprina (Diptera: Calliphoridae) In Different Constant Temperature And Its Significance In Forensic Medicine

Objective: This study concerns the Chronology of development within puparium of Lucilia cuprina (Diptera: Calliphoridae) to determine the postmortem interval (PMI) estimation which is based on the development rates and succession patterns of necrophagous flies and provide fundamental data to forensic entomology.Methods: Flies were collected with an entomological net on a dead rabbit innards from the outdoors.The adults of Lucilia cuprina were bred in laboratory where they were reared successively in a biochemical culture box ,provided with a diet of suger, powdered milk and water,until third generation. Then 70~80 adults were maintained in separate cages with decomposing pork liver to lay eggs.After 2 hours (maximum), eggs were collected to transported to incubators with pork meat as food,where they were maintained under constant temperature(16℃, 20℃, 24℃, 28℃,32℃) at 50~60% RH.The photoperiod and food supply were kept unchanged.The type collection were done when 50% of the larvaes had pupated.10 pupae per sampleing were collected every 12 hours until 50% adults emerged.Keep the specimens in simple fixative at first 24h, then transfer them into 75% alcohol for further preservation.Measurements of pupal weights were made at different temperature and the mean value of each sample of ten pupare was used to establish the growth curves. All the weights data were recorded with the form of Mean±SD.The pupal specimens were observed under a stereomicroscope. Record the regularity of changing colour of puparium with development and make a standard colour plate of the puparium at different temperatures. Under the stereomicroscope ,dissect the pupal case and make minute observation of the morphologic changes according to pupal growth.Try to find some morphological features to be age~markers.Illustrate the chronology of puparia at different constant temperature .Data were analyzed using SPSS11.5 when made statisticalanalysis of the pupal weights.Pupal pictures were disposed with PHOTOSHOP.Result:1 Chronometrical morphologic changes of pupa stagesSeven age-markers of pupal morphological features were collected as follows:Ⅰbody configuration;Ⅱcephalopharyngeal skeleton;Ⅲarchicephalon;Ⅳpupal respiratory horn (PRH);Ⅴartus;Ⅵsetae;Ⅶcompound eye(CE).According to the features ,the pupal period of L.cuprina can be divided into 9 developmental stages: Prepupal stage; Cryptocephalic stage;Phanerocephalic stage;brown thorax setae stage;Tanned thorax setae stage; Tanned abdomen setae stage;Part pigmented eye stage;Entire pigmented eye stage; preadult stage.The results show that some morphological changes within the puparium can be used as age-markers. According to this,the pupal period can be divided into 9 developmental stages. The time required for developing into each stage at different constant temperature was inversely proportional to the temperature, namely, shorter in higher temperature and longer in low temperature.Furthermore,the developing time for each stage lasted inhomogeneously: shorter in both the beginning (Prepupal stage, Cryptocephalic stage)and the near~emergence stage. Phanerocephalic stage took a great part of the entire pupal period. The emergence of pupal respiratory horns( PRH) can beused as a indicator to distinguish Cryptocephalic pupa and Phanerocephalic pupa.At the latter, A pair of pupal respiratory horns was found laterally just prior to the posterior boundary of the first abdominal segment.At Cryptocephalic stage ,the pupal respiratory horn was small,corpora mammillaria,carnation.With pupa growing,this horn slightly protruded from the center of globules on the bubble membrane, corniform, brown.PRH emerging time at different constant temperature was inversely proportional to temperature,at 16℃, 20℃, 24℃, 28℃, 32℃,it respectively is 60h,48h,32h,24h,12h.The appearing of the cephalopharyngeal skeleton on the pupal case can be a marker to determine whether it developed into the second pupal stage,that is Cryptocephalic stage.At prepupa stage, cephalopharyngeal skeleton was tightly fixed in the pupa body,which was extremely resemble with the one of third-instar larvae in both structure and position,so it can not be seen from outside of the puparium.The profile of compound eye(CE)completely formed during Phanerocephalic stage,The colour at this stage was yellow or white yellow which lasted for 4 pupal stages until Part pigmented eye stage.At this time, posterior of the CE initially became bright red,then the red area gradually expanded until Entire pigmented eye stage when the whole eye became cherry .At Preadult stage,CE was crimson.As the observation showed,the yellow stage lasted longer than the pigmented stage.The higher the temperature was,the faster the CE changed.2 Colour of pupariumAt a certain constant temperature,the colour of puparium deepen with developing time.The change was:lighter yellow, yellow,brunneuc,pitchy.Brunneuc maintained the longest time.3 Pupal weightAt a certain constant temperature,it shows gradually lightening in weight with the growing and developing of pupa. The changes was inversely proportional to different temperature, namely, faster in higher temperature and slower in low temperature.Furthermore,the amplitude of lightening was inhomogeneous:deeply at first 24hours, gently for post hours,then sharply at the near-emergence stage.Conclusions: This study demonstrated the necessity of further research in the features of pupal stage which took nearly half part of the immature stage of necrophagous flies.This research mainly referred to Chronometrical morphology, colour of puparium and pupal weight. Based on results from this study, deducing on PMI could be more extensive besides larval stage. For the result were collected under laboratory condititons,it would be more meanful in forensic medicine if experiment was carried on imitating natural environment.