Insects Of Forensic Significance From China And Pakistan With Emphasis On Blowflies And Their Molecular Identification

The present study was aimed at achieving three main goals,to define the urban profile of blowflies,to devise a multiple gene approach for their molecular identification and to examine the association of blowflies and other carrion breeding insects with the Muslim "Festival of sacrifice i.e.Eid-ul-Azha".Community structure of blowflies was determined in the urban settings of Huajiachi, Hangzhou,eastern China.Standard liver-sodium sulfide baits were placed along various urban facilities,including open sewerage drain,garbage dump sites,mulberry plantation, college building,pig farm and meat-vegetable markets.(1) We collected 12 species in all with nine species of major forensic importance including Chrysomya megacephala,Ch. pinguis,Aldrichina grahami,Lucilia sericata,L.illustris,L.porphyrina,Hemipyrellia ligurriens(Diptera:Calliphoridae),Musca domestica(Muscidae) and Parasarcophaga crassipalpis(Sarcophagidae).(2) ANOVA test statistics revealed significant mean population variation between species(F= 251.670) and months(F= 164.231).(3) Ch. megacephala was the only species observed throughout the study year.This species was the most dominant and abundant group with a population mean of 33±3.919 while L. porphyrina with a mean of 0.27±0.122 was the least represented species.(4) A special assemblage of seven species excluding the blowfly Ch.megacephala and housefly M. domestica were found absent in the January of two consecutive years,i.e.2007 and 2008. In February five species L.sericata,L.porphyrina,L.illustris,H.ligurriens and the flesh fly P.crassipalpis were absent in 2007-08 while in March only L.porphyrina and H. ligurriens were absent in 2007-08.Therefore,winter population means of all nine species were considerably low;January=3.3±0.246,February=1.14±0.472,December=0.25±0.76 and LSD analysis showed no significant difference for these months.However, statistical variation was significant when compared for winter and summer months.The second phase of our study dealt with the molecular identification of blowflies collected from Huachiachi.Since blowflies are holometabolous with morphologically different immature stages,the difficulties in the identification of larval stages limit their use as forensic indicators unless there is an alternative.Molecular taxonomy with its modern advances provides that alternative.In this context,molecular sequencing for forensic identification is performed here for the first time for blowflies of Huachiachi,Hangzhou. Five gene segments were specially selected for this purpose from the mitochondrial DNA (mt DNA =COI,CYTB,ND5) and ribosomal DNA(rDNA = ITS1 & ITS2) based on their popular use in forensic genetics.The 75 sequences yielded as a result were submitted to Genbank.We compared inter-specific variation at five gene loci for about 28 pairs of blowfly species(and also blowfly,housefly pairs).Among blowflies,(1) intra-specific sequence variation for ITS1 was in a reasonable range,i.e.0.29-1.16%.On the contrary inter-specific variation was quite high 7.8-57.3%;thus revealing its utility in congeneric species identifications.(2) ITS2 showed an inter-specific variation of 1.2-19%which illustrates a powerful resolution at species level yet at the same time shows an inevitable possibility of wrongful identification for sister species.(3) COI with an inter-specific divergence of 2.2-10.4%also indicated the likelihood of wrongful sister species identification.(4) On the other hand CYTB and ND5 regions with inter-specific divergence at 5.2-15.4%and 4.5-12.4%respectively showed a good resolution power for species level identifications.Among blowflies and housefly pairs(1) ITS2 proved as the most important gene marker for the family level differentiations with 30-35.2%variation.(2) COI with a variation of 10.3-13.5%showed no distinct demarcation between the "blowfly and housefly" pairs and some "blowfly and blowfly" pairs(10.4%).(3) CYTB with an inter-family divergence of 12.5-16.7%also showed no clear separation of species at the family level.(4) However ND5 provided distinct family level resolutions(16-17%).(5) Our results additionally demonstrated the usefulness of a group of concatenated gene segments,i.e.CO1,CYTB,ND5 and ITS2,in blowfly identifications based on phylogeny. (6) We also calculated an evolutionary timeline of blowflies based on our molecular data, previously published GenBank data,contemporary fossil evidence and theoretical assumptions.We conclude that ITS1 as a novel sequence with low intra-and high inter-specific variability shows potential in identification of sister species that are quite similar at other gene loci.ND5 shows potential in identification of geographic populations of blowflies. ITS2 and ND5 both show family level resolution for flies of forensic importance.COI and ITS2 depict the evolutionary break for Calliphoridae and Muscidae at about 20-50 MYA.In a parallel study,which is currently the first of its kind in the world,conducted in Pakistan,we explored insect fauna associated with the festival of Eid ul Azha(Eid).Thus, during the Eid time frame in 2008,in a remote Pakistani village,we examined(a) postmortem insect attractions on various sacrificial products,i.e.blood pools,meat, hides/skins,bones and offal(23 animals),and(b) and insect succession on 11 goat offal at five dump sites.Our results show:(1) Thirty five insect species were collected from 14 sites.(2) Carrion present outdoor attracted a diverse array of first wave of flies both by taxa and in numbers as compared to indoors where the initial wave includes only synanthrops. (3) During the present study we also carried out molecular seqauencing of blowflies for the first time in Pakistan.We sequenced three gene segments(COⅠ,COⅡand ITS2) for the Eid population of blowfly Ch.rufifaciesfrom D.I.Khan city of Pakistan.Additionally we sequenced same gene segments for the Eid population of Ch.rufifacies from Rawalpindi city of Pakistan.Massive transport of animals during the festival time throughout the country might facilitate invasion of forensically important fly species.So far our molecular sequence data for Ch.rufifacies population shows no significant variation for two different cities thus supporting our initial assumption that the Eid festival facilitates geneflow between different regions.Further forensic implications of these results in a broader context of Eid festval are discussed.