Role of House Fly in Determination of Post-Mortem Interval: An Experimental Study in Albino Rats

The potential for contributions of entomology to legal investigations has been known for at least 700 years, but only within the last two decades or so has entomology been defined as a discrete field of forensic science .There are many ways that insects can be used to help in solving a crime, but the primary purpose of forensic entomology is estimating time passed since death. Because blow flies arrive earlier in the decomposition process, they provide the most accurate estimation of time since death. House flies (Musca domestica Linnaeus) are medically and forensically important flies. The aim of this study was to investigate time passed since death according to different stages of house fly life cycle with time variations over months of the year. Materials and methods : 120 mature male albino rats were used, 2 rats were scarified every 6 days and left exposed to houseflies, the different stages of the fly life cycle in relation to different postmortem intervals and represented time were recorded, photographed and statistically analyzed. Results: There is a highly significant statistical difference between the duration of the presence of the different stages housefly life cycle (eggs, 1st, 2nd, 3rd larval instars light colored (1 st stage) and dark colored pupae(2 nd stage) through different months of the year which was shorten in hot months of the year (August (9.6±1.57 h), July (1.5±0.52d), July (3.3±0.4d), August (7.6±0.69d), August (10.4±0.69d) and August (12.3±1.15d) respectively) and much prolonged in cold months of the year (January (27.2±2.85h) , January (3.6±0.51d), January (8.2±1.61d), Feb (14.1±1.52d), January (16.0±1.49d) , and January (21.2±1.22d) respectively). The results showed that time after death could be estimated from certain forms of housefly stage and every stage denote the time with consideration to seasonal temperature state. There is statistically difference between the duration of the presence (p.m. interval) of the different stages of housefly life cycle through different months of the year which was shorten in hot months and much prolonged in cold months. Conclusion: postmortem interval could be determined through the presented forms of the housefly developmental stage in and around the dead body with consideration of presented date of observation.


Introduction
orensic entomology can be defined as the use of insects, and their arthropod relatives, that inhabit decomposing remains to aid legal investigations (Byrd 2010).It is considered the most accurate method in determining the time elapsed since death, particularly when more than 3days have elapsed (Kashyab and Pillai 1989).The house fly is the most common of all domestic flies, accounting for about 91% of all flies in human habitations, and indeed one of the most widely distributed insects, found all over the world from the hot to cold regions near humans or their activities (Wiegmann et al., 2003).
As soon as death occurs, autolysis starts to occur with lysis of cells by digestive enzymes.The body starts decomposing.Bacteria present in the gastrointestinal tract start destroying and fermentation the soft tissue producing liquids and gases like hydrogen sulphide, carbon dioxide, ammonia, methane, sulfur dioxide and hydrogen.The volatile molecules escaping from the decomposing body attract insects (LeBlanc and Logan 2010).Also putrative sulfur-based compounds were found to be responsible for the initial attraction of the flies to the decomposing carcass (Ashworth and Wall 1994) but ammonium-rich F compounds present on the carrion induce the flies to put their eggs or larvae (Joseph et al., 2011).
The adult house fly length range from 6 to 9 mm (Mandal 2015), with the female usually larger than the male.Adults usually live 15 to 25 days, but may live up to two months.Female flies need access to suitable food (protein) to allow them to produce eggs.(Brown et al., 2009).The female housefly usually mates once and stores the sperm for later use, lays batches of about 100 eggs on decayed, fermenting or rotting organic material.(Fasulo 2002) Egg, larva, and pupa are the immature stages of house fly which can be used as entomological evidence for post-mortem interval estimation (Anderson et al., 2004)

Aim of the study
The aim of this study was to investigate time passed since death in experimental albino rats according to different stages of house fly life and correlate this with different with time variations over months along one year from the first of January 2016 to the end of December 2016.

Materials and methods
120 mature albino rat, 2 rats were scarified every 6 days (10 rat per month) by cervical dislocation under inhalation anesthesia and left exposed to houseflies, each dead rat was placed on a layer of soil in a small (30x20x 20 cm) box with wooden floor covered by clear glass plates and the sides were made of perforated nets with pores not allow passage of flies through it.The box was left uncovered until sufficient number of house flies attracted on dead rat then covered.Any other types of flies were discharged.
After appearance of eggs, adult flies were removed from the box to facilitate observation of the different stages of fly life cycle.
The different stages of house fly life cycle were closely observed at different days of different months through the study period and were recorded, photographed and statistically analyzed.
Every larva breeding experiment was photographed every day, starting with their occurrence as the first instar larvae until pupation.Samples of different instars larvae were collected and placed in boiling water to stop their development and allow their measurement.In this way we documented morphological changes during their development.Key developmental stages of each larva with the accurate date and time could be distinguished based on those photographs and by measuring their length.

Statistical Analysis
Data were tabulated and statistically analyzed using Statistical Package for the Social Sciences (SPSS) for windows version 16.0.Data were expressed as mean and standard deviation and Anova test was used, Pvalue of 0.05 or less is considered significant

Eggs
The most common sites where eggs were deposited were among natural orifices like eyes and nostrils, anus, or any small wounds in rat.The time of hatching is vital and so eggs were monitored every few hours.Eggs were white in color about 1.2 mm in length, was laid and piled in small groups (fig7).
There is a statistical significant relation between durations of the presence of housefly eggs over the year; the longest period was in January (27.2±2.85h)followed by February (25.0±3.55 h) and the shortest period was in August (9.6±1.57h) (Table1, and fig.1).

Larvae
The first larvae emerged from the eggs after about early instar larvae are 2 to 5 millimeter (mm) legless, typical creamy whitish in color, cylindrical but tapering toward the head.(fig8), second larvae 5 -10mm long (fig9) and third instar larvae are more than 10mm (fig10).
The present study revealed that statistical significanct difference between durations of the presence of 1stinstar larva in days through different months of the year; the longest period was in January (3.6±0.51d)followed by (3.5±0.52d) in February and the shortest period was in July (1.5±0.52d)(Table 2 and Figure 2).There was a statistical significant difference between the durations of the presence of the 2 nd instar larvae through different months of the year; the longest period was in January (8.2±1.61d)followed by February (8.1±0.87d) and the shortest period was in July (3.3±0.4d)(Table 3 and Figure 3).There was a statistical significant difference between the durations of the presence of the 3rd instar larvae through different months of the year; the longest period was in February (14.1±1.52d)followed by January (13.9±0.73d) and the shortest period was in August (7.6±0.69d).(Table 4 and Figure 4).Pupae Pupae were formed when the last larval skin form a capsule-like case, which varies in color from yellow, red , brown, to black as the pupa ages, complete their development till emerging flies escapes from the pupal cases through a hole on the front of its head (fig11).
Our results revealed that there was a statistical significant difference between the durations of the presence of the light colored stage pupae (from yellow, red to light brown) over the year; the longest period was in January (16.0±1.49d)followed by February (15.7±0.94d) in and the shortest period was in August (10.4±0.69d)(Table 5 and Figure 5).
There is a statistical difference between the durations of development of the dark colored pupae (dark brown and black) over the year; the longest period was in January (21.2±1.22d)followed by February (21.1±1.28d) in and the shortest period was in August (12.3±1.15d)(Table 6 and Figure 6).

Discussion
Arthropods are found in a decomposing vertebrate corpse and these colonizers can be used to estimate time interval between death and corpse discovery at the death scene (Amendt et al., 2004).
Insect life cycles act as precise clocks, which begin within minutes of death.They can be used to closely determine the time of death when other methods are useless.They can also show if a body has been moved after death (Catts and Haskell 1990).
In the current study we searched for eggs in most common sites where eggs were deposited usually in natural orifices like eyes and nostrils, anus, or any small wounds in rat.Eggs were white eggs about 1.2 mm in length, was laid and piled in small groups (Hedges, 2004).The time of hatching is vital and so eggs were monitored every few hours.The findings showed that the duration required for hatching of housefly eggs was dependent on atmospheric temperature in different months of the year.This relation was statistically significant where this duration was much prolonged in January(27.2±2.85h)followed by February and December (cold months of the year) while much shortened in August, July and September (hot months of the year) with least time required for hatching in August (9.60±1.57h).Barnard and Geden in (1993) noted that legless maggot emerges from the egg in warm weather within eight to 20 hours.
The present study revealed that there was a statistical significant difference between the durations of the presence of the 1st instar, 2nd instar and 3rd instar larvae through different months of the year; where the longest period of the 1st instar larvae was in January (3.6±0.51d)followed by February (3.5±0.52d) and the shortest period was in July (1.5±0.52d),2nd instar where longest duration was during January (8.2±1.61d)followed by February (8.1±0.87d) and the shortest period was in July (3.3±0.4d).As regards 3rd instar larvae, the longest period was in February (14.1±1.52d)followed by January (13.9±0.73d) and the shortest period was in August (7.6±0.69d),revealed that larval development was rapid in high temperature during summer months of the year.This is in agreement with (Barnard and Geden 1993) who stated that the larva eat for four days in order to store nutrients for metamorphosis, goes through three instars and a full-grown maggot, 7 to 12 mm long, has a greasy, cream-colored appearance and the optimal temperature for larval development is 35 to 38°C (hot months), though larval survival is greatest at 17 to 32°C.Larvae complete their development in four to 13 days at optimal temperatures, but require 14 to 30 days at temperatures of 12 to 17°C (cold months).Also Sukontason et al. (2008) noted that during the larval stage, the fly passes through three different instars by molting and development of larvae is temperature dependent.
Tullis and Goff (1987) mentioned that by measuring the length or dry weight of the oldest larvae and comparing it with the reference data, age of the larvae can be estimated.
Following the larval phase, they transform into pupae, about 8 mm long, formed from the last larval skin form a capsule-like case, which varies in color from yellow, red, brown, to black as the pupa ages.
Results of the present study revealed that, there was a statistical significant difference between the durations of the development of the 1st stage pupae (light colored) over the year; the longest period from time of death was in January (16.0±1.49d)followed by February (15.7±0.94d) and the shortest period was in August (10.4±0.69d).As regarding the 2 nd stage pupae (dark colored), the longest period was in January (21.2±1.22d)followed by February (21.1±1.28d) and the shortest period was in August (12.3±1.15d).This concluded that pupal development was rapid in high temperature during summer and decreased during low temperature in winter season months of the year.Hogsette and Jacobs (2003) also noticed that Pupae complete their development in two to six days at 32 to 37°C, but require 17 to 27 days at about 14°C.The emerging fly escapes from the pupal case through the use of an alternatively swelling and shrinking sac, called the ptilinum, on the front of its head which it uses like a pneumatic hammer to break through the case and transformation from larva to adult usually takes 2-10 days.Krafsur et al. (1985) showed that adult flies emerge from the pupae in warm climates after only 3-4 days of pupae formation.It can complete its life cycle in seven to ten days ranges from 14 to 90 days under suboptimal temperature.
So knowing the stage of insect inhabitation of corpse and the duration of stages in the insect´s life cycle can lead to an estimate of time since colonization.Estimating the time for insects to find the corpse and start laying eggs allowing the investigator to calculate the post mortem interval.
fig (1) life span of the housefly eggs in hours over different months of the year