Reference no: EM132761350

Age determination is done by comparing the stage of the oldest immature specimens sampled from the cadaver and the environmental conditions, to which it is presumed that they were exposed, with known growth rate data recorded from baseline rearing of Diptera from the same zoogeographical area at a known temperature. The crucial element in the determination of larval age is the progressive increase in total body size (length and weight) with time. The larval instar, and hence approximate age, can be determined by internal morphological analyses. To determine larvae age, it is essential to:

• Measure the larvae size (length and weight), their stage of development (first, second or third instar) and determine whether they are at feeding or post-feeding stage.

• Accurately determine the temperature to which the larvae were exposed during their development.

• Relate the size and feeding stage of the larvae to their age using suitable experimental references or a database of size and stage against age at different temperatures.

The age of immature stages of insects found on a dead body can provide evidence for the estimation of a minimum PMI ranging from 1 day to more than one month, depending on the insect species involved and the climatic conditions at the death scene. Exact species identification of insect samples is the first essential step in estimating the age of the larvae found. Calliphorid flies are known to travel over long distances to find their food source and start the postmortem clock as they visit the dead remains both for oviposition and feeding. Studies have shown that due to their early arrival at a corpse, they are the major sources used by forensic practitioners to provide an accurate estimation of the time since death (Archer and Elgar, 2003; James and Harwood, 1969; Borrer and White, 1970; Bland and Jacques, 1978; Liu and Greenberg, 1989; Hall and Doisy, 1993; Hogue, 1993).

1.4 MAMMALIAN DECOMPOSITION

Decomposition is the gradual breakdown of dead organic matter (Spitz, 1993), which starts within few minutes (depending on various environmental factors, surroundings and situation of corpse) after death and continues over a period (Vass et al., 2002). During decomposition, carcass goes through various physical, chemical and biological changes and releases Volatile Organic Compounds (VOCs) to which insects like blow flies, flesh flies and other carrion feeders are attracted. According to Greenberg, Diptera and Coleoptera represents most of the carrion insects' fauna (Greenberg, 1991).

The insects invade the carrion in a predictable sequence or succession and each group of insects is attracted to dead body at certain stage of decomposition. Some species are attracted to the corpse very shortly after death while others are attracted during active decay and still others visit to the dry skin and bones.

1.5 STAGES OF DECOMPOSITION PROCESS

Various stages of decomposition have been proposed by many forensic entomologists. Bornemissza (1957), worked with dead guinea pigs in Australia and recognized 5 stages of decomposition, namely initial decay (0 to 2 days), putrefaction (2 to 12 days), black putrefaction (12 to 20 days), butyric fermentation (20 to 40 days) and dry decay (40 to 50 days). In studies conducted in Hawaii, USA, five stages of the decomposition have been recognized and these appear to be easily applied to studies conducted in temperate areas (Lord and Goff, 2003). These stages are Fresh, Bloated, Active Decay, Advance decay and Skeletal or Remains (Figure I1). Tomberlin et al. (2011) divided the changes into five stages incorporated into pre-colonization and post-colonization as indicated in figure I2. The five stages of decomposition and insects associated with them are briefly outlined below:

1.5.1 FRESH STAGE

The fresh stage starts after death until bloating of the body begins. This stage has only few noticeable changes associated with corpse like green discoloration of the abdomen, livor mortis, skin cracking, and tache noire may be observed (Goff, 2009). Discoloring of the body is evident due to decomposition of internal organs and accumulation of bacteria. The insect species associated with this stage and the first to arrive on the body are the Calliphoridae, which is then followed by the Sarcophagidae. These insect species feed and lay eggs or larvae on the dead body especially in the natural openings of the head (eyes, nose, ear and mouth), anus and genitals and wounds if any present on the body. During this stage eggs laid by flies starts hatching and feed internally. Depending on weather conditions, it represents 1-3 days of PMI.

1.5.2 BLOATED STAGE

The second stage of decomposition is the bloated stage, where putrefaction starts. During this stage, there is breakdown of the soft tissues of the body. Putrefaction is characterized by the green or purple discoloration of the skin, occurring usually between 36 and 72 hours after death (Dent et al., 2004). Putrefaction results in breakdown of carbohydrates, proteins and lipids into gases, liquids and smaller molecules (Vass et al., 2002). These gases are responsible for the bloating of carcass. This stage is mostly associated with blow fly and maggot mass activity. Maggots metabolic activities results an increase in the internal body temperature. Bloated body's temperature can be significantly above ambient temperature (>50oC) and the body itself becomes a distinct habitat in many ways, independent of the surrounding environment (Goff, 2009). Depending on weather conditions, it represents 2-6 days of PMI.

1.5.3 ACTIVE DECAY/DECAY STAGE

Following the release of the trapped gases, the active decay stage commences. This is when the combined activities of the maggot feeding, and bacterial putrefaction result in the breakdown of the outer layer of the skin and the release of the gasses from the abdomen. Additional compounds, including volatile fatty acids are produced from the continued breakdown of muscle and protein. During this stage body deflates and step into decay stage. Early insect colonizers could be at the post-feeding stage and start migrating away from the resource searching for a suitable place to pupate. By the end of the active decay stage most of the flesh get removed from the body by Diptera larvae.

1.5.4 ADVANCE/POST DECAY STAGE

The next stage of the decomposition stage is the dry stage, where colonization by Diptera cease. Any remaining moist skin and tissue becomes attached to the bone with a leather-like appearance (Vass et al., 2002). This stage is usually comprised of bone, hair, cartilage and skin. Coleoptera particularly, Dermestidae are the dominant species found at this stage. They arrive during later stages of active decay but become dominant during post- decay stage.

1.5.5 SKELETAL/REMAINS STAGE

The final stage is the skeletal stage and at this stage only bones and hairs are present. Carrion-feeding insect activity are mostly absent at this stage. There is little or no smell associated with carcass. This stage may represent PMI of month to years.
LIFE CYCLE OF THE BLOW FLY

Blow flies (Diptera: Calliphoridae) are the first group of the insects that invades a dead body due to the odor of fresh blood. Thus, it is the most important insect when it comes to estimate the time since colonization. Colonization of resource by blow flies can be within minutes of exposure based on factors such as moisture, pheromones, VOC, bacterial assemblage (Tomberlin et al., 2012), other insects and other abiotic factors such as shade. After finding an appropriate place for oviposition, the female blow fly deposits eggs directly on the dead body (Smith and wall,1997), in a position where eggs are protected and in an idle environment (moist environment). This ensure nutrient supply for the hatching process. The life cycle of blow flies includes four stages: egg, larvae, pupae and imago stage (Tao, 1927). During the larval stage, three instars can be separated: 1st, 2nd and 3rd instar, third instar stage is much longer than the first and second and is divided to feeding and post-feeding larvae because of behavioral change. Post-feeding is the stage where larvae stop feeding and migrate away from the food substrate seeking cool, dark and damp conditions in order to pupate. Adult flies emerge after the pupae have undergone metamorphic changes and the cycle begins.

1.7 BIOLOGY OF CHRYSOMYA RUFIFACIES (MACQUART)

The hairy maggot blow fly, Chrysomya rufifacies (Macquart, 1843) is an indigenous species to the Australasian region of Old-World tropics (Norris, 1959; Baumgartner, 1993) and is widely distributed throughout the world. This species was first inscribed from Neotropics in 1978, from a human carcass in Costa Rica (Jiron, 1979). Chrysomya rufifacies was introduced to Central America in 1978 and still increasing its geographic range northward into the southern states of America (Baumgartner, 1993). Chrysomya rufifacies is one of the predominant necrophagous species in central Texas, USA and always arrive spontaneously on a corpse. It is more adapted to tropical condition and found throughout the year (Greenberg and Povolny, 1971). Chrysomya rufifacies larvae (2nd and 3rd instar) are facultative predators and cannibalistic (Baumgartner, 1993). Trials in the laboratory showed that, Chrysomya rufifacies can switch from necrophagy to cannibalism and predating on other blow fly larvae in situations of limited resource (Goodbrod, 1990). Chrysomya rufifacies has a unique life history trait, with females being able to produce offspring of the same sex (Ullerich, 1977). Thus, this makes it difficult to keep colonies of this fly and require greater population size to exclude colonies of all being the same sex.

1.8 BIOLOGY OF CHRYSOMYA MEGACEPHALA (FABRICUS)

Chrysomya megacephala (Fabricius, 1794), the oriental Latrine fly, is widely distributed in the Oriental and Australian regions and adjacent parts (Afghanistan, China, and Japan) of the Palearctic region (Zumpt, 1965; Greenberg and Povolny, 1971; Smith, 1986; Essar, 1991). Its relationship to man varies from hemisynanthropic to eusynanthropic category (Greenberg and Povolny, 1971). The adults of this species are greenish-blue in color and can easily be separated from other calliphorids by the presence of a blackish-brown anterior thoracic spiracle, while in others it is white (Zumpt, 1965; Prins, 1979, 1982).

Another vital character that distinguishes this species from others is that in males the upper two-third of each eye orbit has large facets, sharply distinct from smaller facets of lower one-third. However, in females, all the facets are uniform. The adults have short stout body similar in appearance to Chrysomya rufifacies, but head is larger. The adult flies are attracted to carrion and sweet foods as well as urine and excrement. Although Chrysomya megacephala has noticeable activity during the heat of the afternoon, this species is one of the first species to become active in the early morning hours and the last species to depart carrion at nightfall (Byrd and Castner, 2000). Once the adults have settled on carrion, they are not easily disturbed. Larvae of this species are primarily carrion feeder, and the adults shows preference for fresh cadaver.

1.9 NEW TREND AND DEVELOPMENTS IN FORENSIC ENTOMOLOGY

The use of insect's evidence in forensic investigations requires firstly the identification of the insect species present at the crime scene. This is crucial because closely related insect species that colonize vertebrate carrion can have considerably different developmental rates and hence false identification can lead to inaccurate estimation of PMI. In investigations where immature stages are collected from the crime scene, the forensic entomologist will have to rear the larvae to adult flies. This is only possible if specimens are alive when they reach to the analyst. Due to certain limitations, DNA-based analysis has been suggested by Sperling and co-workers to aid the current identification method as it can be performed at all developmental stages (Sperling et al., 1994). DNA-based techniques have also been studied as a potential for ageing insect species. Tarone and Foran (2011) investigated the gene expression of the blow fly Lucilia sericata during its development to improve age estimation. Zehner and colleagues investigated the potential of using gene expression analysis as an age estimation tool for Calliphora vicina puparia (Zehner et al., 2009). Their results gave age specific expression patterns, allowing for the puparia to be aged in three stages: the beginning, the middle and at the end of their metamorphosis. Hence a method that could be used for identification to the species level at the immature stage as well as ageing without having to rear the insects would be essential and shorten the turnaround time in the estimation of PMI.

1.9.1 AGE GRADING AND SPECIES IDENTIFICATION TECHNIQUES

To establish the PMI, the forensic entomologist aims to determine the age of the oldest colonizing insects' species. However, for the age to be determined the insect species firstly needs to be established. Species identification is challenging in immature stage than in adult form (Byrd and Castner, 2010; Smith, 1986). For species identification and PMI estimation forensic entomologist rear the immature insect's stage till adult flies. Insect rearing at the same temperature and humidity of the surroundings from where the insects were collected is important as these factors have been shown to affect the rate of the development (Marchenko, 2001; Tarone et al., 2011). Also, previous studies have shown that the type of tissue on which the insect species were fed during the developmental process could impact on the size (length and weight) and developmental rate (Clark et al., 2006; Kaneshrajah and Turner, 2004). Muscle attachment site (MAS) found to be a good tool for species identification in case of larval stage.

During this research work, attempts have been made to determine the age of immature stages of forensically important insect species (Chrysomya megacephala and Chrysomya rufifacies) with the help of some recent age estimation techniques. Cuticular hydrocarbon compounds (CHC's) have also been utilized in the field of entomology for ageing, gender identification and species identification. (Tregenza et al., 2000; Savarit and Freveur, 2002; Brown et al., 2000; Desena et al., 1999; Hugo et al., 2006; Brown et al., 1992; Chen et al., 1990; Mpuru et al., 2001; Jackson and Bartelt 1986; Steiner et al., 2006 and Marican et al., 2004). Cuticular hydrocarbons (CHC) may give the same accuracy as DNA techniques for ageing the insects. Composition of cuticular hydrocarbon changes with age in some of the insect's species. These compounds are very useful to age the postfeeding larvae, which is quite long and difficult to differentiate morphologically. Analysis of Volatile Organic Compounds (VOCs) released by insects' stages (immature and mature) can enables the entomologist to distinguish the feeding third instar from post feeding and young pupae from the older one. According to Frederickx and Co-workers, the volatile profile has shown a variation in both composition and quantity in larvae and pupa of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) (Frederickx,2012a).

Gene expression is another alternative approach that is a cost-effective and quantitative measurement to age immature insects.

Research has shown that temporal patterns of gene expression exist throughout immature insect's development (Miller, 1991; Goldsmith and Wilkins, 1995). Dipteran gene expression has been of recent interest in forensic entomology and colleagues have shown that quantitative studies with gene expression can be used to age immature blow flies (Tarone et al., 2007; Zehner et al., 2009; Tarone and Foran, 2011; Boehme et al., 2013, 2014). Such biological information can be used to break lengthier stages (e.g., third instar or pupa) into smaller temporal pieces and improve the precision of insect age estimates. Internal morphological analysis of blow fly pupae by CT-Scan techniques provide additional internal development information to that of external morphological analysis, allowing a more accurate age and thus Postmortem interval estimation. Development of the internal organs inside the pupal shell have the potential of age determination.

How new techniques like VOC, Gene expression, Hydrocarbon composition are cutting the requirement of insect's rearing process to determine postmortem interval?