The corpse is a silent witness who never lies. -Anonymou

Transcription

1 The corpse is a silent witness who never lies. -Anonymou s

2 Forensic entomology, by definition, refers to the association of insects and other arthropods with any legal matter though many people prefer to use this term only as related to the insects associated with a corpse at a murder scene. Application of entomological knowledge during crime investigations requires detailed information about the life history, habits, immature stages, geographical distribution, taxonomy etc. regarding the insects which can be potentially important as forensic indicators. Workers around the globe have laid stress on the importance of entomological evidence and its implications in criminal cases and research papers have continued to pour in dealing with various aspects of the subject. Moreover, seven books dealing exclusively with forensic entomology have been published so far. 1. A Manual of Forensic Entomology by Smith (1986). 2. Entomology and Death: A Procedural Guide by Catts and Haskell (1990). 3. Forensic Entomology- The Utility of Arthropods in Legal Investigations by Byrd and Castner (2000). 4. A Fly For the Prosecution: How Insect Evidence Helps Solve Crimes by Goff (2000). 5. Entomology and the Law: Flies as Forensic Indicators" by Greenberg and Kunich (2002). 6. Maggots, Murder and Men by Erzinclioglu (2002). 7. Forensic Entomology: An Introduction by Gennard (2007). 8. Current Concepts in Forensic Entomology by Amendt, Goff, Campobasso and Grassberger (2010).

3 Overall, there has been a sizeable representation of several hundred research papers dealing with various aspects of forensic entomology. The prestigious Journal, Forensic Science International, published a special issue on forensic entomology in 2002 which included research papers by many renowned forensic entomologists of the world. Similarly, a full volume was devoted to this branch of science in Anil Aggarwal s Internet Journal of Forensic Medicine and Toxicology in Due to non-availability of human bodies for decomposition studies, a wide array of different animal carcasses has been used to simulate humans by various workers from time to time. In most of these studies, insect fauna mainly comprises of Order Diptera (blow flies and flesh flies), Coleoptera (Silphidae, Staphylinidae and Dermestidae) and Hymenoptera. Man has been able to associate insects (mainly flies) with carrion (animal or human) for thousands of years. For example, in Homer s The Illiad, the character Achilles worries about the dead body of his friend, Patrokis, fearing that flies will breed worms and make the body foul (Homer, The Illiad, Book 19). The Illiad is commonly dated back to the 9th or 8th century B.C. The first reference to blow flies was given more than 3600 years ago in the Har-ra-Hubulla, a collection of cuneiform writings on clay. It is one of the oldest known book in zoology and first mentions the green fly and the blue fly (Greenberg and Kunich, 2002). Some 2500 years ago, the Egyptians embalmed deceased individuals in order to protect them from insects and decay. Larvae are even referenced in the Book of the Dead, (Allen, 1960) Chapter 154: That my body will not become prey to larvae.

4 It wasn t until the 13th century A. D. that insects associated with a decomposing tissue was used to solve a crime. In 1247, Sung Tz u, published The Washing Away of Wrongs, a training manual for death scene investigators. In this manual, he tells the story of a murder by slashing that occurred in a rural village of farmers. The wound on the victim appeared to have been made by a sickle, so the investigator had all the men of the village assemble in a line with their sickles lying on the ground before them. The guilty individual was identified when the investigator noticed a cluster of flies surrounding his sickle. Although the weapon had been wiped clean, there was enough blood and other tissue residue left on the blade to attract dozens of flies, thus identifying the suspect (McKnight, 1981). At the beginning of the nineteenth century, Mende (1829) compiled a list of necrophagous insects, including flies and beetles as well as other taxa. Krahmer (1857) described the opportunities and problems associated with using insects for the estimation of the minimum postmortem interval (PMI), many of which are still relevant today. The first application of forensic entomology in a French courtroom, in 1850, can be viewed as a breakthrough for this discipline (Bergeret, 1855). Skeletonised remains of a child were found behind a chimney by workmen during redecoration and insect evidence was accepted as proof that the current occupants of the building could not have been the murderers. At that time, forensic examiners had only a poor understanding of insect biology and their knowledge was based largely on casual observations. Although Weismann (1864) published development data for two necrophagous fly species, it was not widely noted by the forensic community. Yovanovich (1888) and Megnin (1894)

5 were the first forensic examiners who attempted to evaluate insect succession on corpses, properly establishing the science of forensic entomology. The earliest study on this subject in India is believed to be by Mackenzie in Calcutta (Indian Medical Gazette, 1889) where he made observations on dead bodies about the times of appearance of eggs and larvae though not much record is available about this study in the contemporary literature. Johnston and Villeneuve (1897), Leclercq and Leclercq (1948), Leclercq (1968, 1969, 1983, 1997), Nuorteva (1959a, 1959b) and Nuorteva et al. (1974) were among the first to use forensic entomology for the determination of the minimum postmortem interval. Researchers like Reiter and Wolleneck (1982, 1983) and Reiter (1984) hinted about the forensic importance of the common necrophagous fly Calliphora vicina while studying its development. Many others like Marchenko (1980, 1988, 2001), Erzinclioglu (1983a, 1985a, 1986, 2003), Rodriguez and Bass (1983), Vinogradova and Marchenko (1984), Greenberg (1985), Goff et al. (1989), Nishida et al. (1986), Lord et al. (1986),Turner (1987), Introna et al.(1989) and Goff (1991a) have laid stress on the importance of insect evidence during forensic investigations. Now, at the beginning of the 21st century, forensic entomology has been accepted in many countries as an important forensic tool (Keh, 1985 ; Goff, 1991a, 1991b; Greenberg, 1991; Catts and Goff, 1992; Morris, 1994; Anderson, 1996; Benecke,1996 a, b; Introna et al., 1998; Benecke,1998 a,b; Benecke,1999; Bourel et al., 1999; Malgorn and Coquoz 1999; Oliva et al., 1995; Campobasso and Introna 2001; Campobasso et al., 2001; Wolff et al., 2001; Lord and Goff, 2003; Benecke, 2004; Amendt, 2004 a, b). Benecke (2001 b) and

6 Grassberger (2004) described fundamentals and a brief history of forensic entomology. Singh et al. (1999), Anderson (2001a) and Klotzbach et al. (2004) have discussed the development of this science in India, British Columbia (Canada) and German speaking countries, respectively. In order to review the voluminous available literature in a meaningful way, it has been considered better to discuss the relevant papers under different headings. Insect fauna of corpse Hough (1897) studied the fauna of dead bodies with special reference to Diptera and also compared the insect fauna of America with that of Europe. Motter (1898) studied 150 disinterments where dates of original interments were known to him and provided a detailed list of the fauna found on the buried bodies. Fuller (1934) also studied the insect inhabitants of carrion. According to Chapman and Sankey (1955) the dipterous larvae were the only scavengers of any consequence in the decomposition of the carcasses. These provide the basis on which the rest of the fauna, mainly predaceous beetles and parasitic Hymenoptera depend. Bornemissza (1957) studied the effect of carrion decomposition on the soil fauna. This study showed that soil fauna played only a minor role in decomposition of the carcasses. Ants and earwigs were the only members of the soil fauna which fed on carrion. One of the important studies on insects and their relationship to decay rates was undertaken by Reed (1958) on 45 dog carcasses which revealed much information on the ecological process and stages of decomposition. It was reported that the total arthropod

7 populations were greater in summer; however, certain species reached their maximum population during the cooler periods of the year. Burger (1965) studied the succession of sarcophagous Diptera in different seasons of the year on mammalian carcasses. According to Payne (1965) a definite ecological succession occurred among the fauna of carrion. Each stage of decay was characterized by a particular group of arthropods, each of which occupied a particular niche. Payne et al. (1968) studied arthropod succession and decomposition of buried pig carcass. Easton and Smith (1970) pointed out that the appearance and abundance of fauna are related more to the season than to the stage of decay. Payne and Mason (1971) reported eighty two species of Hymenoptera associated with pig carrion. Payne and King (1972), while studying the decomposition of pig carcasses in water, observed that water limited the number and kind of arthropod scavengers reaching the animal carcasses. Wasti (1972) carried out study on common fowl carrion in relation to the incidence and possible succession of arthropods and remarked that in carrion free from arthropods, the rate of decay was very slow and no distinct decompositional stages were recognizable. Lane (1975) studied the blow fly succession on white mice, so as to calculate precisely and accurately the time of death within a period of 4 to 5 days after death during which blow flies were the most significant insects present. Denno and Cothran (1976) studied the competitive interactions and ecological strategies of Sarcophagid and Calliphorid flies inhabiting rabbit carrion. Nuorteva (1977) established that blow flies are attracted to carrion in the fresh stage of decomposition but are not attracted to a carcass when it has progressed to the advanced decay or dry stage. Coe (1978) observed that in

8 the absence of vertebrate scavengers dipterous larvae consume up to 5% of the soft tissue of elephant carcasses, the remaining being utilized by microorganisms. McKinnerney (1978) studied carrion community composition in rabbit carcasses. He described four decompositional stages and collected eighty arthropod species, out of which sixty three were identified as participants in the carrion community. According to Putman (1978) out of all the insects visiting a dead body, the larvae of blow flies and flesh flies are responsible for the maximum consumption of terrestrial carrion. Johnson and Ringler (1979) studied occurrence of blow fly larvae in aquatic habitats i. e. on salmon carcasses and their utilization as food by juvenile fishes. O Flynn and Moorhouse (1979) studied blow fly succession on carrion in order to observe the primary flies of summer and winter seasons and their importance in forensic entomology. Jiron and Cartin (1981) studied decomposition of dog carcasses in Costa Rica and divided it into four stages of decay. Braack (1981) enumerated the visitation pattern of insect species on carcasses in Kruger National Park. The work by Abell et al. (1982) is one of the very few studies made on fauna of reptilian carrion. O Flynn (1983) observed rate of development and succession of blow flies in carrion in Southern Queensland. Lord and Burger (1984) studied the arthropods associated with Herring Gull and Great black backed Gull. According to them, carcasses located in vegetated habitats and hence having less extreme environmental conditions, support significantly higher number of predatory insects, particularly ants, and fewer carrion consumers, thereby slowing carrion consumption and decomposition rate. Smeeton et al. (1984) worked on exposed human corpses. They tried to determine whether certain species were consistently associated

9 with specific time elapsed after death. Lee et al. (1984) reviewed the use of fly larvae from human corpses for estimation of PMI. Goddard and Lago (1985) studied blow fly succession on carrion in order to observe the primary flies of summer and winter seasons and their importance in forensic entomology. Early and Goff (1986) recorded differences of up to 22 C between carcass temperature and ambient air temperature in Hawaiian Islands. They studied arthropod succession patterns in two different habitats i.e. one in xerophytic habitat away from human dwelling and other in wetter habitat (mesophytic) near human dwelling. Smith (1986) described the faunal succession on dead bodies that included exposed, buried, mummified, and burnt bodies. He also discussed how the environmental conditions as temperature, humidity and light influence the fauna on the body. Von Zuben et al. (1987) studied arthropod succession in exposed carrion in a tropical rainforest on Oahu Island.According to Nation and Williams (1989) scavengers can also eliminate insect colonizers on a carcass. Haskell et al. (1989) described the use of aquatic insects to determine submersion interval. Kashyap and Pillay (1989a, 1989b) discussed the role of insects in solving crime. They examined and analyzed sixteen insect infested cadavers to evaluate the reliability of entomological method in estimation of time elapsed since death, in relation to other medico legal approaches. Blackith and Blackith (1990) observed that there were about twenty nine species of insects (excluding Coleoptera) which infest small corpses. Hewadikaram and Goff (1991) compared the effect of carcass size on rate of decomposition and arthropod succession pattern.

10 Schoenly (1992) and Schoenly et al. (1992) conducted a statistical analysis of successional patterns in carrion arthropod assemblages having implications for forensic entomology and its help in determination of the postmortem interval. Isiche et al. (1992) found a difference in the calliphorid species abundance and diversity when observing mouse carrion in sunny and shaded habitats. Four species were found in the sunny area and only two of these species were observed in the shaded area. According to Lee and Marzuki (1993) there are differences in community composition and assumptions could lead to erroneous conclusions pertaining to estimations of time of death. Shean et al. (1993) compared the decomposition rates of two pig carcasses, kept in close proximity to each other, one exposed to sun and the other under shade. El-Kady et al. (1994a) carried out field studies on blow fly and flesh fly succession on exposed rabbit carcasses killed by different causes. They noted that decomposition of carcasses varied according to the manner of killing. They also concluded that the manner of death had induced several noticeable differences in the succession pattern of calliphorids and sarcophagids and in the rate of development of their immature stages. El-Kady et al. (1994b) studied the trophic interactions of carrion-attendant arthropods in a botanical garden in Alexandria, Egypt. Wells and Greenberg (1994a) used goat, rabbit and rat carrion as resource to identify conditions under which native taxa might avoid interaction with the invaders. Dillon and Anderson (1995) present a technical report showing that insects colonize remains in predictable sequence and can be used to assist in determining time of death. Data generated during this work have been used in investigation of six cases. According to Wells and Greenberg (1994b) fire ants, Solenopsis geminata (Fabricius), can have a

11 major impact on necrophagous insects. They found that the colonization of carrion, and in turn the rate of decomposition, was retarded when fire ants were in significant numbers. VanLaerhoven and Anderson (1999) present a report showing that predictable sequence of insect succession occurring on buried carcasses is different from succession on above ground carcasses. Anderson and VanLaerhoven (1996) conducted a practical exercise in forensic entomology as how a study of insect activity on corpses and other crime scene materials can yield valuable evidence. Dillon and Anderson (1996) prepared a database for insect succession on carrion in northern and interior British Columbia while Tantawi et al. (1996) studied arthropod succession on exposed rabbit carcasses in Egypt. Schoenly et al. (1996) conducted a simulation study to quantify statistical uncertainty in succession-based entomological estimates of the postmortem interval in death scene investigations. Keiper et al. (1997) presented a report on midge larvae as an indicator of postmortem submersion interval of carcass. McDonell and Anderson (1997) described the use of aquatic invertebrates to determine the time since submergence. Richards and Goff (1997) studied arthropod succession on exposed carrion in three contrasting tropical habitats on Hawaii Island. Komar and Beattie (1998) studied the decay of clothed pig carcasses of approximate human size and reported that clothing disturbance patterns produced by postmortem insect activity mirrored those associated with a premortem sexual assault. Tomberlin and Adler (1998) made a comparative study on seasonal colonization and decomposition pattern of rat carrion in water and on land in an open field.

12 Bourel et al. (1999) found a considerable difference in blow fly colonization of rabbit carrion from one year to the next in the same location and during the same sample period. De Jong and Chadwick (1999) reported a total of fifty three arthropod species collected from rabbit carrion at different elevations in Colorado. VanLaerhoven and Anderson (1999) observed differences between succession on buried carrion in two biogeoclimatic zones which were chosen on the basis of different soil type and vegetation. Insects are attracted to an animal immediately after death and detection of the remains can be influenced by their presence (Smith, 1986; Bourel et al., 1999; Anderson, 1999, 2000). Carvalho et al. (2000) gave a checklist of arthropods associated with pig carrion and human corpses in Southeastern Brazil. Davis and Goff (2000) compared the decomposition pattern in terrestrial and inter tidal habitats in Hawaii islands. Ma (2000) studied constitution and succession of insect community on pig carcass in Hangzhou while Singh and Bharti (2000) provided a list of forensically important blow flies of Punjab (India). Shalaby et al. (2000) compared patterns of decomposition in a hanging carcass and carcass in contact with soil in a xerophytic habitat on the island of Oahu, Hawaii. LaMotte and Wells (2000) gave p-values for Postmortem intervals from Arthropod Succession Data. P-value obtained is based on likelihood ratio statistic and Fisher s exact test which show comparison of arthropod species present on a mystery carcass to the observed frequency distribution of carcasses exposed to the elements. According to Anderson (2001b) a corpse progresses through a recognized sequence of decompositional stages, from fresh to skeletal, over time. Each of these stages of decomposition is attractive to a different group of sarcosaprophagous

13 arthropods, primarily insects. Singh and Bharti (2001a) enlisted eight species of ants collected from rabbit carcasses during successional studies in the state of Punjab, India. Keiper and Casamatta (2001) and Merrit and Wallace (2001) described importance of benthic organisms as forensic indicators. Joy et al. (2002) compared the larval activity on sunlit and shaded raccoon carrion for a 153h period during the spring. They determined that the maggot mass temperatures in sun and shade-exposed carrion were the same; however, the mean developmental times for P. regina larvae collected from sun-exposed carrion were greater than those shaded carrion. Anderson et al. (2002) determined time of submergence using aquatic invertebrate succession and decompositional changes. Kirkpatrick and Olson (2002) compared the summer succession of necrophagous and other arthropod fauna associated with fresh and frozen pig carcasses. Oliva (2001) and McDowell (2002) described the importance of Diptera in forensic science. Watson and Carlton (2003) reported ninety three arthropod species associated with black bear, deer, alligator, and pig carrion in Louisiana. Bharti and Singh (2003) studied the succession of insect communities on rabbit carrion in the state of Punjab, India. They reported thirty eight species of insects belonging to thirteen families from different stages of carrion decay during various seasons of the year. Grassberger and Frank (2004) studied arthropod succession on pig carrion in central European urban habitat while Grassberger et al. (2003) described Chrysomya albiceps as a new forensic indicator in Central Europe. Iannacone (2003) studied the forensically important arthropod fauna on pig carcass in Peru while Anderson and Hobischak (2004) observed decomposition of marine carrion. Haefner et al. (2004)

14 observed pig decomposition in lotic aquatic system and used algal growth as indicator of PMI. Bourel et al. (2004) studied the entomofauna of buried bodies in France. Carvalho et al. (2004) exposed carcasses of domestic pig in an urban area to determine stages of decomposition and insects of forensic significance exploiting the carcasses. Leccese (2004) sampled several species of forensically important insects using pig meat as bait. Gruner (2004) gave a list of forensically important Calliphorids collected from pig carrion in rural North-Central Florida. Slone et al. (2004) described investigations into the thermal behavior of forensically important larvae, the effect of internal maggot mass heat generation on maggot development time, and the sources and magnitudes of error affecting a phenological computer model that predicted the likely postmortem interval (PMI) of human remains found long after the person s demise. Gill (2005) provided a technical report on decomposition and arthropod succession pattern on pig carrion in rural Manitoba. Morette and Ribeiro (2006) reported the occurrence of Tachinaephagus zealandicus parasitizing pupae of Chrysomya megacephala associated with a decomposing rat carcass in a secondary wood area in Campinas. Vitta et al. (2007) conducted a preliminary study on insects associated with pig carcasses. Five decomposition stages of pig carcasses were categorized: fresh (0-1 day after death), bloated (2 days after death), active decay (3 days after death), advanced decay (4-6 days after death) and dry (7-30 days after death). The arthropod species collected from corpses in the field sites were classified as mainly belonging to two orders and nine families. Heo et al. (2008) studied insect succession on a decomposing piglet carcass placed in a manmade freshwater pond in Malaysia. Wang et al. (2008) studied

15 decomposition of pig carcasses placed in the outdoor environment in different seasons to observe and select the critical entomological index in accurate estimation of PMI. According to Beningern et al. (2008) cadaver decomposition did not result in a significant difference in soil carbon and moisture content. However, significant increases were observed in the concentration of soil ph, total nitrogen, soil-extractable phosphorus, and lipid-phosphorus. According to them there is a significant increase in the concentration of grave soil nutrients that represented a maximum after PMI of 43 days (lipid ), 72 days (total nitrogen), or 100 days (soil-extractable phosphorus). Soil-based method has the potential to act as a tool for the estimation of extended PMI. Matuszewski et al. (2008) studied insect succession and carrion decomposition in various forest habitats of Central Europe. There were no differences between forests in the sequence of insect occurrence on carrion. However, differences between forests in occurrence time and activity period of some taxa were found. Heo et al. (2008) studied insect succession and rate of decomposition on a partially burned pig carcass in an oil palm plantation in Malaysia. They compared the stages of decomposition and faunal succession between a partially burnt pig (Sus scrofa) and natural pig (as control). The burning simulated a real crime whereby the victim was burnt by murderer. Results showed that there was no significant difference between the rate of decomposition and sequence of faunal succession on both pig carcasses. The only difference noted was in the number of adult flies, whereby more flies were seen in the control carcass. Anderson (2008) conducted a study that followed decomposition and animal scavenging on a carcass which helped to explain artifacts and decomposition of a body submerged in

16 deep water. Michaud and Moreau (2009) demonstrated that the occurrence probability of some carrion-related insects on carcasses can be estimated from meteorological records even across seasons with different rates of degree-day accumulation. Gomes et al. (2009) studied insect succession on carcasses decomposing in a sugarcane crop in Brazil. In all seasons, C. albiceps and C. macellaria were frequent visitors during the fresh and bloated stages of decomposition, whereas Dermestes maculatus, Necrobia rufipes and Oxelytrum sp. were characteristic at the most advanced decomposition stages. The fact that climatic variations influence the occurrence of insect species and vegetation in the tropics may help to solve crimes through sampling of the local insect fauna, as may the fact that only certain groups of insects occur in specific regions. Segura et al. (2009) studied succession pattern of cadaverous entomofauna in a semi-rural area of Bogotá, Colombia. They examined the succession of insects colonizing three pig (Sus scrofa) cadavers. In total 5981 arthropods were collected during decomposition, 3382 adults and 2599 immature stages, belonging to 10 orders and 27 families. Voss et al. (2009) study annual, seasonal and shorter term variation in patterns of insect succession on decomposing remains at two contrasting locations in Western Australia. Insect assemblages were strongly correlated between locations, within corresponding time periods, indicating that patterns of insect succession were similar between localised sites within the same broad geographic area. Matuszewski et al. (2009) monitored insect fauna of pig carcasses in different seasons and forests of Western Poland. The composition of carrion fauna and selected features of residency in carrion in

17 adults and larvae of particular taxa were analysed. A total of 131 adult and 36 larval necrophilous taxa were collected. Only 51 adult species and 24 larval taxa were minimally abundant ( 10 specimens) at least on one carcass. Reibe and Madea (2010) investigated the time taken by blow flies to find and oviposit on fresh carcasses placed outdoors and indoors. Paired dead piglets, one in the open and the other in a nearby room were exposed simultaneously on nine occasions. In all cases the indoor piglet carcass was exclusively infested by Calliphora vicina; only in one case, on a very hot day after a 48h exposure did Lucilia sericata infest an indoor carcass. The outdoor piglets were infested by a variety of common corpse-visiting species: L. sericata, L. caesar, L. illustris, C. vicina and C. vomitoria. Oliveria and Vasconcelos (2010) analyzed the occurrence of forensically important insect species (Order Diptera) on fourteen cadavers taken into the Institute of Legal Medicine (ILM), in Pernambuco, Brazil, according to the conditions of the body and the pattern of colonization by insects. Five species were present on cadavers : C. albiceps, C. megacephala and C. macellaria (Calliphoridae), Oxysarcodexia riograndensis and Ravinia belforti (Sarcophagidae). There are so many factors affecting insects succession which include geographic distribution, season, temperature, humidity, habitat, and biology of carrion insects as has been demonstrated in large number of studies (Hanski, 1987; Levot et al. 1979; Hutton and Wasti 1980; Cianci and Sheldon, 1990; Greenberg, 1990; Goff, 1993; Hall and Doisy, 1993; Byrne et al., 1995; Grisbaum et al., 1995; Byrd and Butler, 1996; Johl and Anderson, 1996; Dillon, 1997; Avila and Goff, 1998; DeCarvalho and Linhares, 2001;

18 Arnalds et al., 2001; Anderson and Hobischak, 2002a, 2002b; Archer, 2003; Archer and Elgar, 2003; Archer, 2004 and Tabor et al., 2004). The other method to estimate PMI utilizes the stage of development of the oldest larvae feeding on the corpse, from which one can determine a close approximation of the minimum time since death. Insects often lay eggs within minutes or hs after death (Catts and Goff, 1992) thus providing a developmental reference. According to DeJong (1995) the blow flies can find a dead body within few seconds of exposure. Because most bodies are discovered with in the first few days or weeks, blow flies are encountered more frequently, and can reveal time of death more accurately than their successors (Greenberg, 1991). For estimating the minimum PMI, the age of the available larval stages must be determined. Various procedures for estimating their age exist, but all are based on the fact that the rate of development depends on the ambient temperature. The approach, known as thermal summation (Wigglesworth, 1972), is the accumulation of degree hours (ADH) or degree days (ADD). However, when using estimates of ambient temperature to calculate development rates, it should be noted that these data are not necessarily representative of the temperatures experienced by the insects in the corpse. Large numbers of larvae may create a so-called maggot mass effect which, according to the metabolic and feeding rate of these immature insects, can generate a temperature substantially higher than ambient (Wells and LaMotte, 1995).

19 Life history and morphology of immature stages of blow flies In forensic entomology, information is essential not only about the developmental stages of the insects found on the body, but also on their identity. Morphological methods are usually helpful for this purpose (Schumann, 1971; Smith, 1986; Povolny and Verves, 1997). Wijesundara (1957) studied the life history and bionomics of C. megacephala in Ceylon. Kamal (1958) investigated the effect of controlled temperature and humidity on the life history, rate of development and other biological activity of both adults and immature stages of thirteen species of flies, representing nine genera within the families Sarcophagidae and Calliphoridae. Kitching (1976) described and illustrated the eggs, three larval instars and puparia of C. bezziana. He also compared them with five other species of Chrysomya found in Africa and Oriental region. The most common stage found on the corpse is generally the larva and its identification is a must from forensic point of view. Subramanian and Mohan (1980) studied the life history, bionomics and reproduction strategy of blow flies C. megacephala, C. rufifacies and L. cuprina. Teskey (1981) described in detail the morphology and terminology of Dipteran larvae including those of sarcophagous families. Prins (1982) gave morphological and biological notes on six South African blow flies and their immature stages. Greenberg and Szyska (1984) reared fifteen species of Peruvian blow flies to study the structure of their egg plastrons, larval instars and puparia. They also gave a key to the larvae of known species of Peru apart from studying their developmental rates and diet activity. Erzinclioglu (1985b) provided descriptions based on a detailed comparative study of the structural features of all three larval instars and pupae of the six British Calliphora and single

20 Chrysomya species. Results demonstrated that certain characters, singly or in combination, enable reliable species separation and keys were provided to third instar larvae and pupae of all these species. Erzinclioglu (1987) described diagnostic features of 3rd instar larvae of ten species of blow flies of medical and veterinary importance. He also described characters for recognition of the early instar larvae of genera Calliphora and Lucilia. Erzinclioglu (1988) further described three larval stages of the blow flies, Phormia terranovae, Phormia regina and Borellus atriceps and discussed biology of these flies which according to him were adapted to a cold climate. Liu and Greenberg (1989) provided identification keys and diagnostic descriptions for eggs, three larval stages and puparia, of seventeen species of forensically important flies. Erzinclioglu (1990) studied the larvae of two closely related blow fly species of the genus Chrysomya. Peterson and Newman (1991) studied in detail the chorionic structure of egg of screwworm fly C. hominivorax. Greenberg and Singh (1995) studied eggs of eleven forensically important calliphorid species belonging to six genera with the help of scanning electron microscope for useful diagnostic characters. Amorion and Riberio (2001) studied morphological differences among the puparia of three blow flies species. Results of the study are useful in species identification and from forensic point of view. According to Wallman (2001) the detection of carrion-breeding blow flies provides valuable forensic indicators of time, place and manner of death. Their correct identification is crucial to their successful use in this way. A particular challenge to blow fly identification lies in the morphology or structure of blow fly larvae.

21 Bharti and Singh (2002) reported the occurrence of larval stages of blow flies during different stages of decay in rabbit carcasses. Sukontason et al. (2003a) studied the surface ultra structure of C. rufifacies larvae. Morphological changes were greatest from the first to the second instar, but less from the second to the third instar. Sukontason et al. (2003b) further studied larval morphology of C. megacephala using scanning electron microscope which can help to differentiate C. megacephala from other larvae found in decomposing human corpses. Sukontason et al. (2004b) identified eggs of forensically important fly species using potassium permanganate staining techniques while Sukontason et al. (2004a) differentiated third instars of six forensically important fly species in Thailand with the features of the anterior spiracles, dorsal spines between the prothorax and mesothorax, and posterior spiracles. Chigusa et al. (2006) also identified the species and instar of several species of blow flies. C. megacephala egg ultramorphology was analyzed by David (2008) to generate data for further comparison with other species. Sukontason et al. (2008) studied larval morphology and developmental rate of C. megacephala and C. rufifacies, the two forensically important blow fly species in Thailand. Effect of starvation on larval behavior According to Ullyett (1950) each larva attempts to feed as much as possible to attain optimum size before the food supply is exhausted. Subramanian and Mohan (1980) reported that C. rufifacies attain postfeeding stage after 60-70h of hatching. William and Richardson (1983) studied effect of artificially reduced amount of larval food on life

22 history of L. cuprina, C. stygia, C. vicina and C. hilli. With increasing food shortage, the puparia were reduced upto 12% of weight attained under conditions in which food was unlimited. There is no effect on egg size but reduction in the number of ovarioles. Larval stage is the main period when blow flies face limited food resources. Since these substrates are often booming in insects of various kinds, there is often an intense competition for resources (Hanski, 1987). Goodbord and Goff (1990) studied that with declining food, the immature larvae leave the carrion and search for alternative food resource. During this period if they are unable to get any food source then they enters the period of forced starvation. According to Ives (1991) the substrates (read decaying carrion) in which blow fly larvae develop are discrete and ephemeral. Reis (1994) stated that competition for food is usually of an exploitative type. According to Wells and Kurahashi (1994) C. megacephala attain postfeeding stage after 68 h of hatching at 27 C. According to Campobasso et al. (2001) the fully grown mature larvae search for a place to pupate and disperse away from the carrion. However, if they are not heavy enough to pupate, the larvae may move to another source of food in the vicinity. According to Shiao and Yeh (2008) third instar larvae of C. rufifacies will expel C. megacephala larvae from their foods by using their fleshy protrusion on body surface, and the C. megacephala will be forced to pupate earlier by decreasing their larval growth. Under mixed-species rearing of different ratio, the larval duration of C. rufifacies will increase as the ratio increases, but this result was not exhibited in C. megacephala. According to Ireland and Turner (2006) use of entomological evidence in the estimation of the minimum PMI generally depends on the size and developmental stage of blow fly

23 larvae collected from a corpse. So, the factors which can have an effect on the larval size and growth rate can have implications for reliable minimum PMI determinations. Dispersal behavior of postfeeding larvae First study to consider the burrowing depth reached by fly larvae was done by Travis et al. (1940) who studied lateral migration and depth of pupariation by the larvae of the primary screwworm Cochliomyia americana. Green (1951) studied the behavior of blow fly larvae in the field and observed that they could travel distances of 6 10m from the center of dispersal. Lundt (1964) found that blow flies could be completely excluded by burial under only 2.5cm of soil. Nuorteva (1977) showed that under natural conditions, pupae of several dipteran species can be found directly under an animal corpse or as far away as 6m. Coe (1978) found that for the larvae of two species of Chrysomya feeding on elephant carcasses in Kenya, larvae of C. albiceps preyed on the smaller C. marginalis only in the migratory phase as they left the carcass. Smith (1986) investigated the factors controlling the timing of the exodus of L. cuprina larvae from sheep and carrion at night under field and controlled conditions. This type of analysis is important because it can improve the precision with which the minimum PMI is estimated. Vogt and Woodburn (1982) studied the dispersal of postfeeding larvae of L. cuprina from carrion and found that most of the larvae entered the soil in the immediate vicinity of the host. In addition, most of the larvae sampled were located in the top 2.5 cm of soil with none penetrating beyond a depth of 5cm. The type of host also appeared to

24 influence the subsequent dispersal of the postfeeding larvae. Smith (1986) discussed several important aspects of postfeeding larval behavior associated with the methods and techniques applied in forensic entomology. Empty puparia would indicate that a generation of flies had emerged, although such puparia still contained the third instar mouthparts and spiracles that could be used to identify the fly species. Hanski (1987) described an outdoor cage experiment to prove that localized interactions facilitated the coexistence of species breeding in ephemeral habitats which can affect subsequent search for a suitable pupariation site by the dispersing postfeeding larvae. Greenberg (1990a) compared the dispersal behavior of postfeeding larvae of forensically important fly species in an 8m long channel. The results suggested that postfeeding larvae of Calliphorinae moved farther from the food source, pupated underground, and produced a thinner puparium followed by a longer pupariation period when compared to the larvae belonging to Chrysomyinae. Omar et al. (1994) studied certain behavioral patterns of Malaysian Calliphorid and Muscid species on decomposing cat and monkey carcasses and noted that the late third instar larvae of C. rufifacies, Chrysomya chani, C. villeneuvi, and Ophyra spinigera were the least dispersive compared to Chrysomya megacephala, Chrysomya nigripes, Chrysomya pinguis and Hemipyrellia ligurriens. In addition, predatory behaviour by third instar larvae of C. rufifacies, C. villeneuvi and O. spinigera and cannibalism by C. rufifacies and C. villeneuvi were observed in the field. Godoy et al. (1995, 1996) observed an oscillation in the pupal frequency of C. megacephala and C. putoria as a function of the distance from the feeding substrate.

25 According to Boldrini et al. (1997), these oscillations could be the result of larval aggregations at certain sites in the substrate used for pupariation. Von Zuben et al. (1996) proposed a diffusion model to describe dispersal of larvae that were already buried in the substrate. Bassanezi et al. (1997) fitted the model to the data for the postfeeding larval dispersal of calliphorids obtained by Godoy et al. (1995). Tessmer and Meek (1996) investigated the spatial distributions of Calliphorid pupae based on adult emergence from pig carcasses under field conditions during diffrent seasons. Boldrini et al. (1997) developed a mathematical model to explain the appearance of oscillations during the dispersal of larvae from the food source. Bass (1997) studied the exodus of larvae from pig carcasses on a farm and put forward the view that the larvae left the body to avoid predators and pushed themselves through the soil up to 25 feet away. Kocárek (2001) investigated the postfeeding dispersal of larvae of C. vomitoria and L. caesar from carrion in the field and found that the larvae dispersed exclusively at night, thereby minimizing interactions with diurnal and crepuscular predators. Andrade et al. (2002) used an acrylic channel to examine the influence of predation on larval dispersal in C. albiceps and C. macellaria. Other studies have described general aspects of larval dispersal in a circular arena and have confirmed results obtained using troughs (Gomes et al., 2002, 2003). Unlike the dispersal in only two directions allowed by a trough, a circular arena allows radial dispersal from a central feeding substrate. They concluded that the larvae with the smallest mass tended to move greater distances after food depletion, perhaps in search of new sources of food rather than for a site to pupate. In the wild, postfeeding larval

26 dispersal and the subsequent spatial distribution of pupae at pupariation sites could have implications for the susceptibility of larvae to attack by predators or parasites (Pescke et al., 1987). Because C. albiceps larvae prey on C. megacephala larvae, the latter probably disperse farther and deeper to escape predation (Hanski, 1987; Goodbrod and Goff, 1990; Wells and Greenberg, 1992; Faria et al., 1999; Faria and Godoy, 2001). As shown by Gomes et al. (2003), when both species were considered together, there was a positive correlation between the distances from the food source and the depth of burrowing, which indicated a significant correlation with sample size of larvae dispersing in this study. C. albiceps larvae concentrated closer to the center of the arena and did not have a uniform distribution, whereas C. megacephala larvae had a uniform distribution. This arrangement probably reflected the tendency of the latter species to minimize contact with C. albiceps larvae to avoid predation (Faria and Godoy, 2001). In additional experiments vermiculite and sawdust were used to assess the influence of substrate compactness and density on larval dispersal (Cardoso et al., 1992). Most of the larvae burrowed to a depth of 7 12 cm before pupating, in contrast to the study by Gomes et al. (2003) in which most larvae burrowed to a depth of cm before pupariation. Hence, the type of substrate clearly affected the depth of burrowing but not the pattern of postfeeding larval dispersal. Gomes and Von Zuben (2005) studied some of the most important aspects of larval dispersal in the blow fly C. albiceps by using a circular arena to allow the radial dispersion of larvae from the center. Most of the larvae buried themselves near the center of the arena. Gomes et al. (2006a) reviewed postfeeding larval dispersal in blow flies. Gomes et al. (2006b) studied influence of

27 photoperiod on body weight and depth of burrowing by larvae of C. megacephala. Roux et al. (2006) studied circular dispersal of larvae of P. terraenovae and found the shape of the dispersal to be circular with a concentric distribution around the feeding zone. Gomes et al. (2007) studied combined radial postfeeding behavior of C. albiceps and C. megacephala. According to Arnott and Turner (2008) postfeeding dispersal in blow fly larvae under laboratory conditions is typically very short but may extend for hs or days in the field, because larvae try to find a suitable pupariation site. Lima et al., (2009) performed a computational analysis employing a cellular automata model to investigate the larval dispersal behavior of blow flies. The spatially discrete model developed here, incorporates simple behavioral rules in local interactions to produce the large-scale patterns observed in the larval dispersal process. In particular, oscillations, initially explained as a response to larval aggregation, could be explained by the combination of different mechanisms acting during the dispersal process. An analysis of environmental factors, particularly photoperiod, can be helpful when searching for dispersing larvae around cadavers (Amendt et al., 2004a). The study of blow fly larval dispersal can be of crucial importance in minimum PMI determination because the interval will be underestimated if older dispersing larvae or those that disperse farther, faster, and deeper are not considered (Smith, 1986; Von Zuben et al., 1996; Gomes et al., 2002, 2003; Gomes and Von Zuben, 2004, 2005).

28 Development at different temperatures and correlation between larval age, body length and dry weight Evans (1934) put forward the view that increase in temperature may lead to an increase in the speed of development and, therefore, may have a detrimental effect on the accuracy of PMI calculations. Deonier (1940) studied the relationship of carcass temperature to winter blow fly population and activity and pointed out that after establishment of larvae on carcass, its temperature rises than that of the ambience. Davidson (1944) studied effects of temperature on developmental time of blow fly life cycles and define relationship between temperature and rate of development of insects at constant temperatures. Kamal (1958) investigated effect of controlled temperature and humidity on the life history, rate of development and other biological activity of both adults and immature stages of thirteen species of flies belonging to the families Sarcophagidae and Calliphoridae. He also studied the nutritional aspects of larval forms with special emphasis on how the quantity and quality of food affect life span, rate of growth and size of both individuals and populations with regard to maggot development rate. Payne (1965) reported that carcass temperatures often get elevated due to heat generated by maggot masses and that ambient temperature might not reflect the one to which larvae are exposed. According to Levot et al. (1979) the relative success of each carrion fly species depended upon the ability of the larvae to attain quickly the minimum weight for viable pupariation. Calliphora nociva, Calliphora augur, Chrysomya megacephala and Chrysomya rufifacies were the species best adapted to pupariation at low larval weight i.e., their food requirement for successful pupariation were less than those of other species. Dallwitz (1984) studied influence of constant and fluctuating

29 temperatures on developmental rate and survival of pupae of L. cuprina. Nishida (1984) developed growth tables and growth curves for seven species of blow flies and flesh flies at 15 C, 20 C, 25 C, 30 C and 35 C. On the basis of this study time of molting, pupariation or emergence of adults for each species at different temperatures could be calculated. The stabilizing effect of maggot mass generated heat was studied in human remains by Vinogradova and Marchenko (1984) who also presented threshold developmental temperatures for nine species of blow flies. Williams (1984) developed a computer model for determining the time of blow fly egg hatching based on maggot weight and temperature record. Williams and Richardson (1984) observed that higher temperature of mixed species maggot mass was tolerated by C. rufifacies but the population of L. cuprina was burned out. Introna et al. (1989) studied life cycles of L. sericata reared in the field with continuous registration of temperature, humidity and luminosity. When compared to parallel life cycles after rearing the flies in a growth cabinet, the results showed no statistical difference between the field and laboratory conditions. Goodbrod and Goff (1990) studied effect of population density on rate of development of two species of blow flies. Rearing of C. megacephala and C. rufifacies in pure cultures at seven different population densities (larvae per gram of liver) demonstrated an inverse relationship between density and the duration of the larval stage. According to Block et al. (1990) of all the stages in the life history of blow flies, pupae have the smallest percentage of water compared to body weight. Hagstrum and Milikan (1991) studied differences in insect developmental times between constant and fluctuating temperatures. Catts (1992) discussed the influence of maggot generated heat

30 on corpse community development and on subsequent analysis of entomological data. The level of heat generated in a maggot mass could also selectively affect larvae of different species as recorded by Turner and Howard (1992). Tantawi and Greenberg (1993) and Adams and Hall (2003) checked the effect of killing and preservative solutions and estimates of maggot age in forensic cases. They found that there was shrinkage in various solutions used as preservatives and concluded that larvae killed in boiling water and then placed in preservative solution did not shrink and recommended such treatment of larvae if their length was to be interpreted forensically in a valid and consistent way. Singh and Greenberg (1994) studied survival after submergence in the pupae of five species of blow flies and found that the maximum period of submergence for the pupae after which they could develop into normal adults was four days. These data are potentially useful in estimating duration of submergence of corpse during forensic investigations where the corpse got submerged after pupariation. Wells and Kurahashi (1994) studied the development of C. megacephala at 27 C and observed that there were differences in the length of postfeeding larvae and attributed these differences to their sensitiveness to environmental conditions. Wells and LaMotte (1995) estimated maggot s age from its weight using inverse prediction and also constructed a confidence interval on age of larva, given its weight. Johl and Anderson (1996) studied the effect of refrigeration of immature stages on the development of Calliphora vicina at a temperature of 3 C for 24h and concluded that such a treatment of any stage (egg, larva, and pupa) induced a 24h delay in adult emergence. Milward-de-Azevedo et al. (1996) checked the influence of temperature on