Forensic genetic analysis of insect gut contents

REVIEW Forensic Genetic Analysis of Insect Gut Contents Carlo P. Campobasso, PhD, MD,* Jason G. Linville, PhD,† Jeffrey D. Wells, PhD,‡ and Francesco Introna, MD* Abstract: Entomological evidence is most often used for estimating the postmortem interval, but fly larvae can also be a source of vertebrate DNA. Forensic analysis of DNA recovered from a larva’s gut can be used to identify what the larva had been feeding on. During our previous research studies, we used the same DNA extraction for the dual purpose of identifying the insect species and associating a maggot with its last meal. In our experience, we have encountered several situations where this method for associating a maggot with a corpse would have been useful, such as removal of remains from a suspected crime scene, an alternative food source is nearby the scene or the body, and a chain-of-evidence dispute. However, since maggot gut content analysis is a quite brand-new area of study, many of the limitations of the technique have not yet been explored. The results of our most recent research studies suggest that third-instar larvae actively feeding on the corpse can be considered the best source of human DNA, better than postfeeding or starved larvae. In this paper, the state of the art of forensic genetic analysis of maggot gut contents is reviewed. Key Words: forensic science, forensic entomology, DNA analysis, maggot crop (Am J Forensic Med Pathol 2005;26: 161–165) M aggots are commonly used during a death investigation to estimate the period of insect activity based on aging the oldest insect specimen associated with the corpse.1,2 In estimating the postmortem interval, it is assumed that all of the maggot’s development and feeding period occurred on the corpse since usually the larvae are collected directly from or near the corpse. However, such a direct assumption cannot always be made. In our experience, we have encountered Manuscript received October 13, 2004; accepted March 14, 2005. From the *Section of Legal Medicine, University of Bari, Bari, Italy; the †Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama; and the ‡Department of Biology, West Virginia University, Morgantown, West Virginia. Reprints: Carlo P. Campobasso, PhD, MD, Section of Legal Medicine, University of Bari, Piazza Giulio Cesare, Policlinico, Bari, Italy. E-mail: [email protected]. Copyright © 2005 by Lippincott Williams & Wilkins ISSN: 0195-7910/05/2602-0161 DOI: 10.1097/01.paf.0000163832.05939.59 several situations where an alternative method for associating a maggot with a corpse would have been useful, such as removal of remains from a suspected crime scene, an alternative food source is nearby the scene or the body, and a chain-of-evidence dispute.3 We successfully recovered both human and animal host DNA from the crops of necrophagous fly larvae. The same DNA extraction served the dual purpose of identifying the insect species and associating a maggot with its last meal.4 Most animal molecular-systematic techniques rely on the mitochondrial DNA (mtDNA) molecule.5,6 Due to the high copy number per cell and maternal inheritance, mitochondrial DNA analyses provide a very useful tool for the identification of human tissue that is degraded or that requires comparison to a maternal relative.7–15 When detecting host DNA in the insect alimentary canal, primer specificity is essential.16 Identification of humans usually relies on 2 particularly hypervariable regions (HVI and HVII) within the noncoding control region or D-loop.17–22 Vertebrate animal species determination may also be based on the D-loop; however, for most animals the locus of choice is a protein-coding gene (eg, cytochrome B, Cyt B) for vertebrates23–25 and cytochrome oxydase subunits 1 and 2 (COI⫹II) for insects.26 –31 Vertebrate DNA has been successfully amplified and analyzed from several insect sources. DNA recovered from insect blood meals has been used to identify the species or individual identity of the host.32,33 Gokool et al34 detected human DNA from mosquito blood meals using microsatellites referred as variable numbers of tandem repeats (VNTR). Minisatellites were used to detect human DNA in the mosquito blood meal up to 26 hours after ingestion,35 demonstrating that DNA isolated from mosquitoes is qualitatively and quantitatively sufficient for DNA typing. In a forensic context, human DNA has been isolated and amplified from crab-louse blood meals36 and crab-louse excreta.37 This may be useful in a forensic investigation to identify individuals involved in certain cases of body violence because a louse can be transferred from assailant to victim during a sexual assault. MtDNA has been also typed from beetle larvae recovered from human bones subjected to environmental exposure for several months,38 thus demonstrating that mtDNA may survive late-stage decompositional events and The American Journal of Forensic Medicine and Pathology • Volume 26, Number 2, June 2005 161 Campobasso et al The American Journal of Forensic Medicine and Pathology • Volume 26, Number 2, June 2005 be subsequently recovered even from late-succession insects. Genetic analyses of insect gut contents may prove to be crucial evidence to link a suspect to the victim or the scene, reconstruct the circumstances of the crime, or establish the credibility of the statements made by witnesses. Maggot Gut Content Analysis and Wash Methods As previously demonstrated,3,4 Diptera larvae (also called maggots) can be a suitable source of human mtDNA. Such analyses have focused on the crop, an anterior portion of the gut that forms a food-storage organ. It must be certain that the extracted DNA comes from the gut and not from possible contamination on the maggot’s exterior. In fact, exterior contamination would interfere with making correct inferences about whether a maggot had been feeding on alternative or multiple food sources (human body, animals, etc) and even on the identity of the missing person. For this reason, it is strongly advisable to provide DNA extraction of the crop rather than the entire maggot, also because removal of an internal structure preserves taxonomically important cuticular surface structures and because the crop contents are relatively undigested compared with those of the stomach or intestine. The method usually followed for maggot dissection has been illustrated in detail by Linville and Wells.39 It includes that the 2 or 3 most posterior segments of the maggot are usually cut off with iris scissors followed with a ventral incision made from the posterior to the anterior end of the maggot. During this incision, the scissors must be kept just under the cuticle to prevent damaging the crop. Following the incision, the cuticle can be folded back exposing the viscera, and the crop can be easily removed with forceps. In their research study, the above authors39 demonstrated that before dissection of the maggot and removal of its crop, washing maggots with 20% bleach solution is an effective method for sterilizing the external surface of a blow fly larva, thus reducing significantly any kind of external contamination without any interference with crop mtDNA analysis. Some maggots were intentionally contaminated by soaking them in cow blood for 3 hours and then washed by using 3 different methods (DNase enzyme, distilled water, and 20% bleach solution). Based on the PCR and sequencing results, only washing the maggots in 20% bleach reduced significantly the amount of vertebrate DNA amplified from the maggot’s exteriors. From these experiences, several more questions arose about the preservation method’s effect on genetic analysis, the persistence of recoverable vertebrate DNA in blowfly larvae throughout their development, and the digestion time of DNA in maggots after their removal from the food source. 162 Preservative Conditions and Genetic Analysis of Host DNA Since maggot gut content analysis is a new area of study, many of the limitations of this technique have not yet been explored and we tried to investigate several of them. For example, the method of maggot preservation may affect the investigator’s ability to successfully extract vertebrate DNA from the maggot’s crop. In fact, the type of preservation fluid can change the physical characteristics of the maggot, which may inhibit the investigator’s ability to dissect the maggot and remove the crop intact. This assumption is consistent with the observations made by Tantawi and Greenberg,40 who demonstrated a severe shrinkage effect of several killing and preservative solutions in maggots. Also, the chemical properties of the preservation fluid may inhibit the extraction of DNA from the crop. Formalin-containing solutions are commonly used to preserve the external identifiable features of entomological evidence, but formalin has been known to degrade high-molecular-weight DNA41 and inhibit the extraction of DNA from formalin-fixed tissues.42 Regarding the ability of storage temperature and type of preservation fluid to alter the stability of human or other vertebrate DNA within the maggot crop, preliminary results suggest that maggots should be stored and preserved as soon as possible in ethanol or at a low temperature.43 Using 8 preservative conditions on maggots dissected after different time intervals (2 weeks, 8 weeks, and 6 months) it was demonstrated that ethanol should be considered a better preservative solution at refrigeration or room temperatures since other kind of solutions such as formaldehyde or Kahle’s solution can seriously prevent host DNA analysis over time.43 In maggots preserved in 95% ethanol at room temperature, although the dehydrated crop became attached to other internal organs and often broke during dissection, this did not prevent the recovery of host DNA. In maggots preserved in formaldehyde, although the crop was easily removed, quantitation results suggest a reduced amount of DNA had been extracted, in most cases preventing the amplification of the HVII region. Maggots stored without any preservation fluid degraded over time at both refrigeration and room temperatures. However, the best results were observed in maggots stored at ⫺70°C without any preservation fluid.43 Maggot’s Development and Digestion Time of Host DNA Another factor the investigator should consider during analysis is the maggot’s stage of development, as the relative size and condition of the entire maggot and its crop may affect the strategy of dissection and extraction. The size of the maggot and its crop may render different strategies for extracting vertebrate DNA from the gut contents. In fact, young maggots may be too small for dissection and crop removal. In older postfeeding maggots, the larvae stop feed© 2005 Lippincott Williams & Wilkins The American Journal of Forensic Medicine and Pathology • Volume 26, Number 2, June 2005 ing and the crop contents are emptied into the remainder of the maggot gut. Alternative methods of analysis, such as extraction of the entire maggot, may provide better results for maggots that are too small for dissection or for postfeeding maggots when the crop is no longer visible. In this matter, we investigated the degradation of host DNA during the maggot development and after a maggot is removed from the food source. The purpose was to answer the 2 main questions dealing with genotyping host DNA: when can human DNA be recovered throughout a maggot’s development? How long can a maggot cease feeding before gut content DNA cannot be recovered? In the attempt to answer to the above questions, maggots of Calliphora vicina were collected at half-day intervals for 6 days: one group was immediately preserved, and some other maggots were kept alive off the food source for 24 hours and for 48 hours. Preliminary results suggest that mtDNA was successfully amplified from most of the groups of maggots collected in different stages of development that had been immediately preserved and some kept 24 hours off the food source.44 DNA was not recovered from any maggots that had been removed from the tissue and kept alive for 48 hours.45 DNA sequencing failed in most maggots less than 2 days old. Maggots 2–2.5 days old were too small for dissection, but extraction of the entire maggot did allow for the recovery of human DNA. In older postfeeding maggots with near-empty crops, extraction of the intestines failed to recover human DNA. These data are consistent with those illustrated by Dadour et al,46 who were able to detect ingested host DNA throughout all stages of the life cycle of Calliphora dubia (Diptera: Calliphoridae), including first-stage larvae at day 1 and pupae at day 2, that is, approximately 24 hours after the pupal case has formed and the larva has stopped feeding and, therefore, comparable to 24 hours postfeeding period of Calliphora vicina. In fact, no DNA was amplified in pupae of C dubia at day 3, consistent with 48-hour postfeeding period of C vicina maggots or kept alive for so long. However, in our results STR analysis was successful only for maggots 2.5– 4.5 days (fully third-instar larvae) immediately preserved. Young and postfeeding individuals, as well the group of maggots kept alive for 24 hours and 48 hours, failed to produce a STR genotype at any locus as shown in Table 1. Quantiblot results revealed that the total human DNA recovered from this latter group of maggots was very low, falling below the detection limit of 0.06 ng/␮L (Linville et al, unpublished data). We believe that the cause of the sporadic mtDNA success and failed STR attempts is essentially due to the amount of food stored inside the gut content, as clearly expressed by the crop size. In C vicina third-instar larvae, the crop is commonly 7 mm long (one third of the total maggot length) at the peak feeding, but just a day later (24 hours), after the crop is emptied into the gut, it is reduced to 3 mm long.47 In our experience, all maggots © 2005 Lippincott Williams & Wilkins Insect Gut Contents TABLE 1. Host mtDNA and STR Results of Maggots Immediately Preserved and Kept Off the Food Source for 24 and 48 Hours Age Less than 2 days Young third instar (2–2.5 days) Fully third instar (2.5–4.5 days) Postfeeding (4.5–6.5 days) Less than 2 days Young third instar (2–2.5 days) Fully third instar (2.5–4.5 days) Postfeeding (4.5–6.5 days) Less than 2 days Young third instar (2–2.5 days) Fully third instar (2.5–4.5 days) Postfeeding (4.5–6.5 days) Alive, Hours Crop/ Intestine mtDNA STR 0 0 No/no Yes/yes No Yes No No 0 Yes/yes Yes Yes 0 Yes/yes Yes No 24 24 No/no No/no No No No No 24 Yes/yes Yes No 24 No/no No No 48 48 No/no No/no No No No No 48 No/no No No 48 No/no No No whose crops were approximately 1 mm or less did not produce consistent results. In fact, in postfeeding maggots 48 hours after peak feeding, the size remains about the same, approximately 1 mm long. Therefore, the smaller crops of postfeeding maggots, as those of young maggots, reduce the chance of successfully amplifying DNA and obtaining a genetic profile. Another factor affecting DNA analysis to be considered is the lipid deposition that usually occurs in maggots towards the end of the third instar since postfeeding larvae show high lipid concentrations. In this respect, removal of the maggot crop should be always preferred over extraction of the entire maggot for genetic analysis of insect gut contents. Regarding biochemical alterations of DNA during food digestion, there does not appear to be any severe enzymatic breakdown of host DNA in the crop over time. Primarily digestion does not occur within the crop, because proteolytic enzymes are not secreted into this area of the foregut, which mainly acts as a food container. However, enzymes are present in the saliva of the maggot for preoral digestion and are reincorporated with the food into the crop.48,49 Thus, some degradation of DNA certainly occurs within the crop, but it seems to be not the main reason to a failure of DNA typing from the crop material. Zehner et al50 have recently demonstrated that also the time of storage of the maggots and 163 Campobasso et al The American Journal of Forensic Medicine and Pathology • Volume 26, Number 2, June 2005 the length of the postmortem interval up to 16 weeks appeared to have no particular influence on the quality of DNA results. CONCLUSION Based on the above preliminary results, we do not imply that amplification of DNA from small crops is impossible since DNA laboratories better equipped for low-level DNA analysis could get better results from young and postfeeding maggots or even from pupae, as demonstrated by Dadour et al.46 It seems for sure that some other laboratories can certainly get better STR results very useful for investigators; for example, to determine the identity or the sex of the individual on which the maggots feed. This is consistent with the results reported by Clery,51 who detected prostate-specific antigen (PSA) and obtained a male Y-STR type from maggots colonizing a cadaver in a simulated postmortem sexual assault; PSA was recorded from sonication of whole postfeeding larvae after 145 hours while correct Y-STR profiles were recorded from the crops of actively feeding secondinstar larvae after 48 hours of initial semen deposition. Also Zehner et al50 performed STR typing and HVR amplifications using crop contents of maggots collected from corpses after various postmortem intervals. Our results suggest that host DNA analysis is possible in maggots fully developed and actively feeding on the corpse but is not as likely in older postfeeding or starved maggots with empty crops approximately 1 mm long or less (see maggots kept off the food source for 24 – 48 hours). Since crop contents greatly decrease within 24 hours off the food source, it is crucial to preserve maggots immediately at the crime scene in proper preservative conditions (ethanol or at low temperatures). A morphologic study of the crop (color and length) before gut-content analysis could provide useful information for genotyping host DNA, as well for aging the maggots, as suggested by Greenberg.47 It must be taken into consideration that crop length can vary, depending on larval development, as well as on the species; for example, the crop of P sericata empties rapidly during the first day after peak feeding, while the crop of C rufifacies empties gradually. Therefore, since degradation of DNA within the crop, time of storage of the maggots, and length of the postmortem interval do not seem to influence DNA typing, based on the results illustrated above and relevant literature examined, we believe that the crop size is the main factor affecting the ability to analyze host DNA in maggots and the success of the genetic analysis. Actually, we are trying to expand our experience, and future work is scheduled to analyze vertebrate DNA from maggots that have been collected on different food sources, as well as from different species of Diptera. 164 REFERENCES 1. Catts EP. Analyzing entomological data. In: Catts EP, Haskell NH, eds. Entomology and Death: A Procedural Guide. Clemson, SC: Joyce’s Print Shop; 1990:124 –135. 2. Goff ML. Estimation of postmortem interval using arthropod development and successional patterns. Forensic Sci Rev. 1993;5:81–94. 3. Introna F Jr, Wells JD, Di Vella G, et al. Human and other animal mtDNA analysis from maggots. Proceedings of the 51th annual meeting, American Academy of Forensic Sciences (AAFS). Orlando, 1999:196 (abstract G74; oral presentation). 4. Wells JD, Introna F Jr, Di Vella G, et al. Human and insect mitochondrial DNA analysis from maggots. J Forensic Sci. 2001;46: 685– 687. 5. Avise, JC. Molecular Markers, Natural History and Evolution. New York: Chapman & Hall; 1994. 6. Avise JC. Phylogeography: The History and Formation of Species. Cambridge, MA: Harvard University Press; 2000. 7. Holland MM, Fisher DL, Mitchell LG, et al. Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War. J Forensic Sci. 1993;38:542–553. 8. Piercy R, Sullivan KM, Benson N, et al. The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int J Legal Med. 1993;106:85–90. 9. Gill P, Ivanov PL, Kimpton C, et al. Identification of the remains of the Romanov family by DNA analysis. Nat Genet. 1994;6:130 –135. 10. Wilson MR, DiZinno JA, Polanskey D, et al. Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med. 1995;108:68 –74. 11. Holland MM, Fisher DL, Roby RH, et al. Mitochondrial DNA sequence analysis of human remains. Crime Lab Digest. 1995;22:109 –115. 12. Ivanov PL, Wadhams MJ, Roby RK, et al. Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nat Genet. 1996;12: 417– 420. 13. Lutz S, Weisser HJ, Heizmann J, et al. mtDNA as a tool for identification of human remains: identification using mtDNA. Int J Legal Med. 1996;109:205–209. 14. Holland MM, Parsons TJ. Mitochondrial DNA sequence analysis: validation and use for forensic casework. Forensic Sci Rev. 1999;11:21–50. 15. Morley JM, Bark JE, Evans CE, et al. Validation of mitochondrial DNA minisequencing for forensic casework. Int J Legal Med. 1999;112:241– 248. 16. Hogendoorn M, Heimpel GE. PCR-based gut content analysis of insect predators: using ribosomal ITS-1 fragments from prey to estimate predation frequency. Mol Ecol. 2001;10:2059 –2067. 17. Anderson S, Bankier AT, Barrel BG, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981;290:457– 463. 18. Aquadro CF, Greenberg BD. Human mitochondrial DNA variation and evolution: analysis of nucleotide sequences from seven individuals. Genetics. 1983;103:287–312. 19. Horai S, Hayasaka K. Intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrial DNA. Am J Genet. 1990;46:828 – 842. 20. Sullivan KM, Hopgood R, Gill P. Identification of human remains by amplification and automated sequencing of mitochondrial DNA. Int J Legal Med. 1992;105:83– 86. 21. Miller KWP, Dawson JL, Hagelberg E. A concordance of nucleotide substitutions in the first and second hypervariable segments of the human mtDNA control region. Int J Legal Med. 1996;109:107–113. 22. Lutz S, Weisser HJ, Heizmann J, et al. Location and frequency of polymorphic positions in the mtDNA control region of individuals from Germany. Int J Legal Med. 1998;111:67–77. 23. Kocher TD, Irwin DM, Wilson AC. Evolution of the cytochrome b gene of mammals. J Mol Evol. 1991;32:128 –144. 24. Zehner R, Zimmermann S, Mebs D. RFLP and sequence analysis of the cytochrome b gene of selected animals and man: methodology and forensic application. Int J Legal Med. 1998;111:323–327. 25. Bataille M, Crainic K, Leterreux M, et al. Multiplex amplification of mitochondrial DNA for human and species identification in forensic evaluation. Forensic Sci Int. 1999;99:165–170. © 2005 Lippincott Williams & Wilkins The American Journal of Forensic Medicine and Pathology • Volume 26, Number 2, June 2005 26. Sperling FA, Anderson GS, Hickey DA. A DNA-based approach to the identification of insect species used for postmortem interval estimation. J Forensic Sci. 1994;39:418 – 427. 27. Zhang DX, Hewitt GM. Insect mitochondrial control region: a review of its structure, evolution and usefulness in evolutionary studies. Biochem Sys Ecol. 1997;25:99 –120. 28. Malgorn Y, Coquoz R. DNA typing for identification of some species of Calliphoridae: an interest in forensic entomology. Forensic Sci Int. 1999;102:111–119. 29. Vincent S, Vian JM, Carlotti MP. Partial sequencing of the cytochrome oxydase b subunit gene I: a tool for the identification of European species of blow flies for postmortem interval estimation. J Forensic Sci. 2000;45:820 – 823. 30. Wells JD, Sperling FAH. Commentary on: Sperling FA, Anderson GS, Hickey DA. A DNA-based approach to the identification of insect species used for postmortem interval estimation. J Forensic Sci. 1994; 39:418 – 427; and on Vincent S, Vian JM, Carlotti MP. Partial sequencing of the cytochrome oxydase b subunit gene I: a tool for the identification of European species of blow flies for postmortem interval estimation. J Forensic Sci. 2000;45:820 – 823. J Forensic Sci. 2000;45: 1358 –1359. 31. Wells JD, Sperling FAH. DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae). Forensic Sci Int. 2001;120:110 –115. 32. Coulson RM, Curtis CF, Ready PD, et al. Amplification and analysis of human DNA present in mosquito blood meals. Med Vet Entomol. 1990;4:357–366. 33. Boakye DA, Tang J, Truc P, et al. Identification of bloodmeals in haematophagous Diptera by cytochrome B heteroduplex analysis. Med Vet Entomol. 1999;13:282–287. 34. Gokool S, Curtis C, Smith D. Analysis of mosquito blood meals by DNA profiling. Med Vet Entomol. 1993;7:208 –215. 35. Kreike J, Kampfer S. Isolation and characterization of human DNA from mosquitoes (Culicidae). Int J Legal Med. 1999;112:380 –382. 36. Lord WD, DiZinno JA, Wilson MR, et al. Isolation, amplification, and sequencing of human mitochondrial DNA obtained from human crab louse, Pthirus Pubis (L.), blood meals. J Forensic Sci. 1998;43:1097– 1100. 37. Reploge J, Lord WD, Budowle B, et al. Identification of host DNA by amplified fragment length polymorphism (Amp-flp) analysis: preliminary analysis of human crab louse (Anoplura: Pediculidae) excreta. J Med Entomol. 1994;31:686 – 690. 38. DiZinno JA, Lord WD, Collins-Morton MB, et al. Mitochondrial DNA sequencing of beetle larvae (Nitidulidae: Omosita) recovered from human bone. J Forensic Sci. 2002;47:1337–1339. © 2005 Lippincott Williams & Wilkins Insect Gut Contents 39. Linville JG, Wells JD. Surface sterilization of a maggot using bleach does not interfere with mitochondrial DNA analysis of crop contents. J Forensic Sci. 2002;47:1055–1059. 40. Tantawi TI, Greenberg B. The effect of killing and preservative solutions on estimates of maggot age in forensic cases. J Forensic Sci. 1993;38:702–707. 41. Tokuda Y, Nakamura T, Satonaka K, et al. Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde. J Clin Pathol. 1990;43:748 –751. 42. Shedlock AM, Haygood MG, Pietsch TW, et al. Enhanced DNA extraction and PCR amplification of mitochondrial genes from formalin-fixed museum specimens. Biotechniques. 1997;22:394 –396, 398, 400. 43. Linville JG, Hayes J, Wells JD. Mitochondrial DNA and STR analysis of maggot crop contents: effect of specimen preservation technique. J Forensic Sci. 2004;49:341–344. 44. Linville JG, Campobasso CP, Hayes J, et al. Amplification of mitochondrial DNA and STR loci from maggot crop contents throughout the maggots development. In: The recovery and characterization of vertebrate DNA from forensically important fly larvae: an optimization study 关dissertation兴. Birmingham (AL), University of Alabama at Birmingham; 2003. 45. Campobasso CP, Linville JG, Introna F. Potential forensic utility of genotyping human DNA from maggot gut contents: how long can a maggot be off the corpse and still be a useful source of human DNA? Proceedings of the First Meeting of the European Association for Forensic Entomology (EAFE). Frankfurt (Germany), 2003; pp 27 (abstract OC21; oral presentation). 46. Dadour IR, Roenterdink E, Carvalho FC. To determine the rate of fly development for more accurate postmortem intervals: to equip investigators with an array of tools using fly larvae. Proceedings of the XIX Congress of the International Academy of Legal Medicine (IALM). Milan (Italy), 2003; pp 165 (abstract OP109; oral presentation). 47. Greenberg B. Flies as forensic indicators. J Med Entomol. 1991;28:565– 577. 48. Hobson RP. Studies on the nutrition of blow-fly larvae II. J Exp Biol. 1932;9:128 –138. 49. Hobson RP. Studies on the nutrition of blow-fly larvae III. J Exp Biol. 1932;9:359 –365. 50. Zehner R, Amendt J, Krettek R. STR typing of human DNA from fly larvae fed on decomposing bodies. J Forensic Sci. 2004;49:337–340. 51. Clery JM. Stability of prostate specific antigen (PSA), and subsequent Y-STR typing, of Lucilia (Phaenicia) sericata (Meigen) (Diptera: Calliphoridae) maggots reared from a simulated postmortem sexual assault. Forensic Sci Int. 2001;120:72–76. 165