Insect succession associated with corpse ' s decomposition of the guinea pig Cavia porcellus in Benha city , Egypt

Eleven domestic guinea pig corpses were employed as models for studying decomposition and insect succession in Benha city, Egypt, from April 2009 to March 2010. Ambient temperature, faunistic succession over time, and the rate of decay in different seasons were all compared. Results indicated that ambient temperature is the chief factor determining the seasonal variations in decay rate. The diversity of insect community increased as the state of decomposition advances. Members of Dermistidae and Forrmicidae were the first coleopteran and hymenopteran colonizers in all seasons. Sarcophaga, Wholfortia and Chrysomya was observed in spring and summer. Meanwhile, species of Wholfortia were absent in winter and Crysomya species were absent in fall. Patterns of insect succession occurred in a predictable sequence that varied across different seasons. The rate of corpse's decomposition was faster in summer and autumn as compared to spring and winter


INTRODUCTION
Insect colonization of corpses has been demonstrated to occur in a predictable sequence Payne (1990), Rodriguez & Bass (1983), Early & Goff (1986), Goff (1993), Anderson & Van Laerhoven (1996), Van Laerhoven & Anderson (1999) and Tabor et al., (2004).The specific period of colonization of certain insects on corpses can be established as forensically significant.Insects are either attracted to specific products of decomposition or are predators on these necrophagous insects Smith (1986), Catts & Haskell (1990) and Anderson (2001).If the sequence of insect succession on corpse is known for a given geographical region and a specific set of variables, it can be compared against collected species from bodies of unknown time of death to yield the postmortem interval, provided circumstances are similar Anderson (2001).
However, intervals based on succession patterns require knowledge of insect fauna in the geographic region in which the Corpse is discovered, as species vary widely with geographic region (Anderson and Van Laerhoven 1996).Ambient temperature, season, and microclimate of the postmortem habitat also play major roles in the determination of the invertebrate assemblage on corpse (Smith, 1986 andCatts &Haskell, 1990).Several researchers have examined the differential effects of season on necrophagous fly activity (Introna et al., 1989 andChen et al., 1991), decomposition (Mann et al. (1990) andDe Carvalho &Linhares (2001) and insect succession (Archer & Elgar, 2003).These investigators concluded that season has a major effect on the invertebrate assemblage discovered on corpse and the time of insect colonization.Thus, it is crucial to examine seasonal insect activity on corpse in specific geographic regions and various habitats within these regions.
Locally generated data on arthropod succession and development increases the precision of postmortem interval estimations Goff (1993).The present study was designed for studying decomposition and insect succession of domestic guinea pig under the environmental conditions of Benha city in different seasons.Results of the present work may provide entomological data that can be employed in forensic cases in Benha and other similar biogeoclimatic regions.

Study site
The study was conducted in a walk-in insectary, in the entomological lab, Faculty of science, Benha University (30°27'34"N 31°11'8"E).This lab was designed to mimic a normal room in a home.

Experimental animals
Guinea pigs of a relatively uniform size were chosen to simulate the soft skin of a new baby.
On the delivery day of each season, guinea pigs were weighed, and then euthanized with air injection to mimic the normal death case without any chemicals or drugs.After death, the animals were immediately delivered postmortem to the research site in a covered plastic box and prepared for placement at the chosen study site.Then, animals were immediately placed into plastic cages and left exposed to natural conditions.Each Guinea pig was placed on its side within 1 h of the time of death.A tray containing sawdust was placed under each cage to facilitate the collection of larvae, leaving corpses for pupation.

Field protocols
Experimental protocols were modified from that of Anderson & Van Laerhoven (1996).The experiments have been modified for cost-effectiveness, without sacrificing validity.Thus, two, three or four guinea pig corpses were employed per season (3 experiments in spring, 4 experiments in summer and 2 experiments in both autumn & winter).All precautions were made to keep corpse disturbance to a minimum during sampling.Observations and sample collections were made daily at varying times according to each experiment.
During collection days, representative samples of immature and adult insects were collected from and around the corpse.While all insects observed were sampled, there was a definite focus on flies and beetles.
Adult flies were collected with an aspirator and preserved as it is in a glass vials for identification.Flies were labeled as teneral adults if the cuticle was relatively pale and soft compared to the mature adult.Adult beetles, immature insects and other hard-bodied crawling insects were collected by hand or with forceps and immersed in 70% alcohol.
For each corpse, approximately 20 larvae were collected from every distinct maggot mass on the body.Approximately half of the specimens collected were preserved in a glass vials while the other half were kept alive for rearing.The live specimens were placed in jars containing a piece of beef liver.The jars were covered with paper towel, secured with rubber bands and left in the same lab.
All samples were labeled with the date and time of collection, the corpse number, the sampling site from the corpse of and the stage of development at the time of collection.
The temperatures were obtained from the nearest weather station.

Analyses
Excel sheet was employed to compare between the maximum, minimum and mean temperature for each season separately.
For each excel sheet, the mean temperatures were derived from the average of every temperature reading on an hour-basis for a 24-h period from 12 am to 11:59 pm each day.

RESULTS
Eleven laboratory experiments were performed to monitor insect succession and rate of decomposition of guinea pig corpses at room conditions in different seasons of the year.
Although the processes of decomposition and insect invasion were continuous, they were often described by discrete stages, which were characterized by the insect activity at each point in decomposition.
The processes of decomposition were divided into following stages: 1) Fresh stage.A corpse is considered to be in the fresh stage from the moment of death until the first signs of bloating.This is the stage during which the blow flies (Calliphoridae) and flesh flies (Sarcophagidae) arrive at the corpse and begin laying eggs or larvae (Fig. 1, a). 2) Bloating stage.This stage marks the beginning of putrefaction.Anaerobic bacteria produce gases as the result of metabolic processes, which cause bloating.Bloating usually occurs first in the abdomen, although the corpse may later assume a fully inflated appearance (Fig. b).
3) Active decay stage.This stage begins when gasses escape and the remains deflate.During this stage, dipteran larvae forming large maggot masses were predominant.
Large numbers of coleopterans also begin to arrive.By the end of the decay stage, most of the flesh has been removed from the corpse and most of the Calliphoridae and Sarcophagidae have departed from the remains to pupate (Fig. c).4) Advanced decay stage.The coleopterans (particularly dermestids) usually are the predominant species during this stage when the remains have been reduced to skin, cartilage, and bones (Fig. d).5) Dry stage.In this stage, the remains consist of only of hair and bones.Most of the previous taxa will have disappeared, leaving mainly mites as the useful indicators of the PMI during this stage (Fig. e).

Spring experiments Decomposition of corpse
The defining characteristics of each stage of decay, along with the associated ambient temperature readings are summarized in Table (1).In spring, Fresh stage began with death and ended when bloating was initiated.Fresh stage lasted from day 0 to day 2 for all corpses.The beginning of the bloating stage was in 3 rd day and continued to 7 th day.Active decay stage lasted 6 days from 8 th day to 13 th day.Advanced stage was observed in the 14 th and 15 th day.Dry stage began with the 16 th day and lasted till the 37 th day (Table 2).

Insect succession
Table (3) showed the insect succession of forensically important insects on the corpse during spring season.
Larvae and adults of Diptera and Coleoptera as well as Formicidae adults were observed on the corpse throughout the experimental time.Flies appeared from the 1 st day as adults and continued their development in all decomposition stages of the corpse.Five dipteran species were observed during the experiment (Musca domestica, Sarcophaga carnaria, Wholfortia magnefica, Drosophila melanogaster and Chrysomya albiscepes).
Sarcophaga carnaria was the most abundant fly throughout the experiment.All its developmental stages (larvae to adults) were observed on the corpse.It appeared as adult from the 2 nd day (Fresh stage) to the 4 th day (Bloating stage).They also visited the corpse occasionally in both days 17 and 25 (Dry stage).Sarcophaga larvae were collected from the 4 th day (100%) (Bloating stage) to the 9 th day (92.86%)(Active decay stage).Its pupae were observed at the 10 th day (Active decay stage) to 29 th day (Dry stage).Adult emergence of Sarcophaga was recorded at the 27 th day to 29 th day (Dry stage).Adult Chrysomya albiscepes appeared at the first 4 days of the experiment (Fresh and Bloating stages).Also, they visited the corpse occasionally at day 36 (Dry stage).Chrysomya larvae were collected only in 6 th day (7.04%)(Bloating stage).
Drosophila melanogaster appeared on the corpse as adult from 5 th day (Bloating stage) to 37 th day (Dry stage).Musca domestica were observed as adults in 3 different stages of corpse decomposition (Fresh, Bloating and Dry stages).Wholfortia magnefica were observed as larvae only from 3 rd day (Bloating stage) to 9 th day (Active decay stage).Two coleopteran species were observed during the experiment (Attagenus pictus and Dermestes castaneus).Attagenus pictus was the most abundant beetle throughout the experiment.Its adults and larvae were observed on the corpse.It appeared as adult from 5 th day (Bloting stage) to 37 th day (Dry stage).Attagenus larvae dominated itself (100%) from 18 th day to 37 th day (Dry stage), Wherase, Dermestes castaneus were observed on the corpse as adult from 5 th day (Bloating stage) to 9 th day (Active decay stage) and from 16 th day to 20 th day (Dry stage).

Summer experiment Decomposition of corpse
The defining characteristics of each stage of decay, along with the associated ambient temperature readings are summarized in Table (1).
In summer, Fresh stage began with death and ended when bloating was initiated.Fresh stage lasted from day 0 to day 1 for all corpses.The beginning of the bloating stage was in 2 rd day only.Active decay stage lasted 2 days (3 rd and 4 th day).Advanced stage was observed in the 5 th and 6 th day.Dry stage began with the 7 th day and lasted till the 23 th day (Table 2).

Insect succession
Table (4) showed the insect succession of forensically important insects on the corpse during summer season.
Larvae and adults of Diptera and Coleoptera as well as Formicidae adults were observed on the corpse throughout the experimental time.Flies appeared from the 1 st day as adults and continued their development in all decomposition stages of the corpse.Four dipteran species were observed during the experiment (Musca domestica, Sarcophaga carnaria, Wholfortia magnefica.and Chrysomya albiscepes).

Sarcophaga carnaria
were observed on the corpse as adults, larvae and Pupae.Sarcophaga appeared as adults from the 1 st day (Fresh stage) to the 3 rd day (Active stage).Sarcophaga larvae were collected from the 3 rd day (53.19%)(Active stage) to the 7 th day (100%) (Dry stage).Its pupae were observed at the 6 th day (Advanced decay stage) to 20 th day (Dry stage).Adult emergence of Sarcophaga was recorded at the 16 th day to 20 th day (Dry stage).Wholfortia magnefica were observed on the corpse as adults, larvae and Pupae.Wholfortia were observed as adults in the 1 st day only.Its larvae were collected from the 3 rd day (46.81%)(Active stage) to the 6 th day (29.17%)(Advanced stage).Its pupae were observed at the 6 th day (Advanced decay stage) to 21 th day (Dry stage).Adult emergence of Wholfortia was recorded at the 15 th day to 21 th day (Dry stage).Chrysomya albiscepes were observed on the corpse as eggs, larvae and adults.Adult Chrysomya appeared at the first 3 days of the experiment (Fresh, Bloating and Active stage).Chrysomya eggs were collected in the 2 nd day (Bloating stage).
Larvae were observed from the 4 th day (28.26%) (Bloating stage) to 6 th day (14.58%)(Advanced stage).Its pupae were observed at the 6 th day (Advanced decay stage) to 15 th day (Dry stage).Adult emergence of Chrysomya was recorded at the 15 th day (Dry stage).
Musca domestica appeared at the first 3 days of the experiment (Fresh, Bloating and Active stage).
Two coleopteran species were observed during the experiment (Attagenus pictus and Dermestes castaneus).Attagenus pictus was the most abundant beetle throughout the experiment.Its adults and larvae were observed on the corpse.It appeared as adults from 2 nd day (Bloting stage) to the end of the summer experiment (Dry stage).Attagenus larvae dominated itself 100% from 13 th day to 23 th day (Dry stage).Whereas Dermestes castaneus were observed on the corpse as adults from 2 nd day (Bloating stage) to 7 th day (the beginning of Dry stage).One Formicidian species was observed during the experiment (Monomorium phoraensis).It appeared only as adults during the 1 st two days (Fresh stage).Also, they were observed again from 7 th day (the beginning of Dry stage) to 23 th day (Dry stage).

Fall experiment Decomposition of corpse
The defining characteristics of each stage of decay, along with the associated ambient temperature readings are summarized in Table (1).
The rate of decomposition resembled that in the summer except that advanced decay prolonged one day than summer.This stage lasted 3 days from (5 th day to 7 th day).Also, dry stage prolonged 21 day from (8 th day to 28 th day) (Table 2 and Fig. 6).Larvae and adults of Diptera and Coleoptera as well as Formicidae adults were observed on the corpse throughout the experimental time.

Insect succession
Flies appeared from the 3 rd day as eggs and larvae and continued their development in all decomposition stages of the corpse.Three dipteran species were observed during the experiment (Sarcophaga carnaria, Wholfortia magnefica and Chrysomya albiscepes).Sarcophaga carnaria were observed on the corpse as adults, larvae and Pupae.Sarcophaga larvae were collected from the 5 th day (70.59%)(Advanced stage) to the 11 th day (90.91%)(Dry stage).Its pupae were observed at the 8 th day (Dry stage) to 22 th day (Dry stage).Adult emergence of Sarcophaga was recorded at the 18 th day to 22 th day (Dry stage).Wholfortia magnefica were observed on the corpse as adult, larva and Pupa.Wholfortia larvae were collected from the 5 th day (29.41%)(Advanced stage) to the 11 th day (9.09%)(Dry stage).Its pupae were observed at the 8 th day (Dry stage) to 22 th day (Dry stage).Adult emergence of Wholfortia was recorded at the 20 th day to 22 th day (Dry stage).Chrysomya albiscepes were observed on the corpse as eggs, larvae and adults.Chrysomya eggs were not collected or observed but its pupae were observed at the 8 th day (Dry stage) to 14 th day (Dry stage).Adult emergence of Chrysomya was recorded at the 12 th to 14 th day (Dry stage).

Winter experiment Decomposition of corpse
The defining characteristics of each stage of decay, along with the associated ambient temperature readings are summarized in death and ended when bloating was initiated.Fresh stage lasted from day 0 to day 5 for all corpses.The beginning of the bloating stage was in 6 th day and continued to 10 th day.Active decay stage lasted 13 days from 11 th day to 23 th day.Advanced stage was observed from the 24 th till the 28 th day.Dry stage began with the 29 th day and lasted till the 50 th day (Table 2).Insect succession: Table (6) showed the insect succession of forensically important insects on the corpse during winter season.Larvae and adults of Diptera and Coleoptera as well as Formicidae adults were observed on the corpse throughout the experimental time.
Flies appeared from the 2 nd day as adults and continued their development in all decomposition stages of the corpse.Four dipteran species were observed during the experiment (Musca domestica, Sarcophaga carnaria, Drosophila sp and Chrysomya albiscepes).Sarcophaga carnaria were observed on the corpse as adults, larvae and Pupae.Sarcophaga larvae were collected from the 8 th day (100%) (Bloating stage) to the 20 th day (100%) (Active decay stage).Its pupae were observed at the 17 th day (Active decay stage) to 48 th day (Dry stage).Adult emergence of Sarcophaga was recorded at the 42 th day to 48 th day (Dry stage).Chrysomya albiscepes were observed on the corpse as eggs, larvae and adults.Chrysomya eggs were collected in the 4 th & 5 th day (Bloating stage).Larvae were observed from the 12 th day (5.26 %) to the 16 th day (12.5%)(Active stage).Its pupae were not observed.Drosophila sp.appeared on the corpse as adult from 2 nd day (Fresh stage) to 38 th day (Dry stage).They were reobserved in the 46 th day.Its larvae were observed from the 8 th day to the 12 th day (Bloating stage).Musca domestica were observed as adults from the 4 th day (Fresh stage) to the 9 th day (Bloating stage).
One coleopteran species was observed during the experiment (Attagenus pictus).It appeared as adults from 26 th day (Advanced stage) to 48 th day (Dry stage).Attagenus larvae were observed from 39 th day to 48 th day (Dry stage).
One Formicidian species was observed during the experiment (Monomorium phoraensis).It appeared only as adults during the all stages of corpse decomposition.
Accidentally, Spodoptera littoralis appeared on the corpse in the 6 th day (Bloating stage).

DISCUSSION
Five species of adult flies belonging to four dipteran families were collected from corpses throughout the four seasons: Chrysomya albiceps (Calliphoridae), Sarcophaga carnaria (Sarcophagidae), Wholfortia magnifica Calliphorid and Sarcophagid flies were the first colonizers to breed on guinea pig corpses.This finding was consistent with the results of other studies in different geographic areas (Smith, 1986;Monteiro-Filho & Penereiro, 1987;Anderson &Van Laerhoven, 1996 andHall, 2001).
Our results clarified that flies showed an oviposition preference for natural body openings (mouth, nose, and anus) and also hairy areas of the body.This may be due to the high moisture and lower intensity of light.The preference of flies in these areas for oviposition was also observed by Norris (1965).
The present study showed that Sarcophaga carnaria was the most important component of insect succession on guinea pig corpse during all seasons because it was the most abundant species in all experiments.Convenient results were presented by Denno and Cothran (1975).In addition, larvae of Sarcophaga carnaria were collected in all seasons, larvae of Wholfortia magnifica were collected in summer, spring and autumn, larvae of Chrysomya albiceps were collected in summer, spring and winter and larvae of Drosophila melanogaster were collected in winter only.It is worthy to mention that not all species visited the corpse to lay eggs or larvae.Musca domestica was found visiting, copulating, and feeding on the substrate or using it as an extension of their habitat.This observation was convenient with Dear (1978) andDe Souza &Linhares (1997).
Although Sarcophagid species were coexisting with Chrysomya albiceps on the same corpse, Chrysomya albiceps, was responsible for minimum guinea pig corpse consumption.These results confirmed results presented by Tantawi et al. (1996).Contrary to our results, Early and Goff (1986) reported that the numbers of sarcophagid maggots were much less than those of Calliphoridae.Consequently, calliphorid flies ranked second regarding the reduction of corpse weight.
Insect colonizers on the corpse could be separated into four ecological categories.The first category, including the greatest number of necrophagous individuals that fed directly on the corpse is of the highest significance in determining PMI.This includes species in the families Calliphoridae, Sarcophagidae and Dermestidae.Predators and parasites of the necrophagous species comprised the second category.Among the predators of particular significance were larvae of Chrysomya albiceps.Chrysomya albiceps larvae were not only fed on the corpse but also were reported as predators on other larvae infesting the corpse.These results are consistent with the results presented by Tantawi et al. (1996) and Pérez et al. (2005).This explains the occurrence of dead larvae of Sarcophaga carnaria and Wholfortia magnifica near the corpse throughout the experiments.
The third category consisted of the omnivorous species, Formicidae ants as Monomorium phoraensis and Camponotus maculatus that fed on both corpse and associated arthropods.The fourth category was comprised of incidental or adventives species having no direct relationship to the corpse as cotton leafworm, Spodoptera littoralis.These results generally agree with those documented by (Payne, 1965).
Regarding corpse decomposition in relation to fly activity, calliphorid and sarcophagid flies (1 st colonizers) played a fundamental role in corpse decomposition.Delaying of insect infestation resulted in significantly retarded and incomplete corpse decomposition (Payne, 1965 andAnderson &Van Laerhoven, 1996).This could explain why the corpse took more time for decomposition in winter rather than other seasons.Composition and abundance of the corpse-related fauna was interpreted regarding the influence of temperature (Goddard &Lago, 1985 andTantawi et al., 1996).
Season and corpse microenvironment are also factors influencing the species composition and successional patterns during decomposition (Hanski, 1987).In comparison to other studies done in spring and summer season in relation to the diversity number.The collected arthropods species were less in diversity than other authors (Reed, 1958;Rodriguez & Bass, 1983;Lord &Burger, 1984 andArnaldos et al., 2004) in similar seasons.Such contrast agrees with Tentawi et al., (1996);Galal et al., (2009) andChittaro et al., (2005) who collected only 4-5 species during the hot summer.Therefore we could assume that the high temperature, which had been also recorded in the current study, had accelerated the decomposition process, meaning that the corpse is reduced to bones in a shorter time period leading to rapid depletion of food resource and reduction of arthropod colonization time.

CONCLUSIONS
This investigation demonstrated that the patterns of decomposition and insect succession varied across different seasons (Fig. 6).Ambient temperature was a critical factor in the determination of the rate of decay in various seasons (Table 1).
Furthermore, the seasonal distribution of insects' significantly impacted the species that were recovered from corpse in different times of the year.Several families of insects arrived in a predictable sequence, although the pattern varied in different times of the year and in different habitats.
Although a large number of insect species were observed at corpses, relatively few used the corpse for breeding purposes.This indicates that species differ in their ability to use the various resources provided by the corpse.Generally, the first fly species to colonize the corpse had an advantage over later arriving taxa, and their larvae had a greater chance to develop to the adult stage.Both the Calliphoridae and Sarcophagidae demonstrated a preference for the dark putrefaction stage of decomposition (Stage III), although they were present in early and late stages.Other coleopteran taxa, such as members of the Dermistidae and Scarabaeidae dominated in later stages of decay, although their presence and colonization times varied across season.
Further research is needed on the biological and ecological characteristics of the particular species associated with corpses in the Benha Ecozone.The data generated from this research are now available for homicide investigations in Benha and Similar biogeoclimatic regions.

Fig. 6 :
Fig. 6: Comparison of decay rates of guinea pig Cavia porcellus porcellus in different seasons of the year 2009/ 2010 in Benha city, Egypt.

Table 1 :
Ambient temperatures during decomposition stages of the guinea pig Cavia porcellus porcellus in different seasons of the year 2009/ 2010 in Benha city, Egypt.

Table 2 :
Decay rates of the guinea pig Cavia porcellus porcellus in different seasons of the year 2009/ 2010 in Benha city, Egypt.

Table 3 :
Occurrence of forensically important insects collected from guinea pig carrion in the Spring, 2009 of Benha city, Egypt.

Table 3 :
Continued.It appeared only as adult during the 1 st two days (Fresh stage).Also, it was observed again from 7 th day (the end of Bloating stage) to 28 th day (Dry stage).

Table 4 :
Occurrence of forensically important insects collected from guinea pig carrion in Summer, 2009 of Benha, Egypt.

Table 5 :
Occurrence of forensically important insects collected from guinea pig carrion in the Autumn, 2009 of Benha, Egypt.

Table 6 :
Occurrence of forensically important insects collected from guinea pig carrion in the Winter season, 2010 of Benha, Egypt.

Pupa of different flies (100%) Insect succession associated decomposition of the guinea pig C. porcellus in Benha city 13Table 6 :
Continued.