A study of insect succession of forensic importance: Dipteran flies (diptera) in two different habitats of small rodents in Riyadh City, Saudi Arabia

2.1 Study area

The study area is located in Al-Dir'iya, Riyadh, Saudi Arabia. Al-Dir'iya is a town located west of Riyadh and is a semiarid, urban area. The climate of Al-Dir'iya is hot and dry in summer, with cool winters. The present experiment was conducted in winter for 40 days, from January 14 to February 23, 2019. Albino rat and mice carcasses were placed in two different study areas, which are approximately 1.5 km apart; the botanical gardens (surrounded by shrubs and trees) and 12 m above the ground on the roof of a building which is covered with cement and clear of any vegetation. Both sites are located in King Saud University campus in Riyadh, Saudi Arabia (24° 43′ 19.2″ N, 46° 37′ 37.2″ E), the temperature in the study sites were recorded daily (Table 1).

2.2 Experimental design

Twelve carcasses were used in this study (six albino rats and six mice were killed by cervical dislocation), divided equally into two groups. The first group was placed in the botanical garden, while the second group was placed on the building roof. Each rat and mice weighed 300 g and 40 g respectively. At each location, carcasses were then placed in a plastic boxes covered with steel wires and some weight was provide above each cage to keep cages constant and protect carcasses from the attack of large animals 15 m away from each other. Insects attracted to the animal carcasses were collected daily for one hour from 13:00 to 14:00. During insect collection, the climatic conditions were monitored in the field using a hygro-thermometer (model MM 5202-Incoterm™.(The mean temperature in all stages of decomposition was 17.73° (Table 1).

Adult flies were collected as per the method described by Mashaly and Al-Mekhlafi (2016). The collected flies were preserved in 96% ethanol after washing them in tap water, and then transferred in labelled tubes to the laboratory. Preserved specimens were kept at 4 °C for further use.

2.3 Morphological identification

The collected samples were examined under stereomicroscope (SMZ18; Nikon-Japan), and identified following morphological identification key of Marshall et al. (2011). A part of the collected samples was preserved and deposited in the entomology laboratory in zoology department at King Saud University. After the morphological examination, some of the collected insects were stored in 96% ethanol in preparation for molecular identification.

2.4 DNA extraction and polymerase chain reaction (PCR)

From 49 randomly selected samples, Mitochondrial DNA (mt DNA) was extracted using a QIAamp DNA Mini Kit (50) (QIAGEN, Germany), following the manual instructions. The extracted DNA was then preserved at −20 °C.

A partial sequence of the mitochondrial gene COI was amplified using the Cyto1-primers 5′-GGTCAACAAATCATAAAGATATTGG-3ʹ(forward) and 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ (Folmer et al., 1994).

PCR was performed in a 20 μl reaction volume, containing 2.0 μl of DNA template, 0.6 μl of each primer, 4.0 μl of 5 × FIREPol® Master Mix (Solis Biodyne, Spain) (Reagents, FIREPol® DNA polymerase, 5 × Reaction Buffer B [0.4 M Tris-HCl, 0.1 M (NH₄)₂SO₄, 0.1% w/v Tween-20], 12.5 mM). Targeted sequence were amplified using thermal cycler (Applied Biosystems By Thermo Fisher Scientific- USA) under the flowing conditions: an initial denaturation step at 95°c for 15 min followed by a 35 cycles each cycle consist of denaturation at 95°c for 45 s, annealing at 41°c for 45 s and extension at 72 °c for 45 s. A final extension step at 72 °c for 10 min was added. With the help of ethidium bromide staining (Vivantis Technologies, Malaysia), the PCR products were electrophoresed and visualized using 1% agarose gel (Haddadi et al., 2019).

2.5 Sequencing of DNA and data analysis

For molecular identification of collected species, DNA sequencing was performed at the DNA Sequencing Unit of Macrogen (Seoul, Korea), a precision medicine and biotechnology company. Obtaining sequences were edited using Sequencher v4.0.5 software (Gene Codes Corp., United States), identified using the Basic Local Alignment Search Tool (BLAST; National Centre for Biotechnology Information [NCBI]) (Samerjai et al., 2020).

Regarding to insect succession and number of collected specimens (form each identified species), the Obtained data were represented as mean ± SE and subjected to one-way (ANOVA) tests. All statistical analysis procedures were carried out with SPSS statistics 26 software (SPSS Inc., Chicago, IL); when p-values were ≤0.05, differences between means were considered significant.

3.1 Carcass decomposition

The carcasses of both mice and rats undergo four decomposition stages: fresh, bloated, decay, and dry remains, determined by the physical changes to carcasses (Figs. 1 and 2; Table 2). The mean temperatures (both minimum and maximum) recorded during each stage are displayed in Table 1. The decomposition process for rat carcasses lasted for an average of 27.66 and 39.66 days on the building roof and in the botanical garden, respectively. However, for mice carcasses, decomposition lasted for an average of 14.75 and 26.33 days on the building roof and in the botanical garden, respectively.

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Fig. 1

Stages of rat, Rattus norvegicus, decomposition: (A) Fresh, (B) Bloat, (C) Decay, and (D) Dry.

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Fig. 2

Stages of mice, Mus musculus, decomposition: (A) Fresh, (B) Bloat, (C) Decay, and (D) Dry.

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3.2 Insect succession on carcasses in different stages of decomposition and habitats

The mean number of flies recovered from the different decomposition stages of rat and mice carcasses at the two study sites was 1,748, representing 11 families and 14 species. Insect number and species composition differed between decomposition stages and habitats. The results of the statistical analysis showed significant variations in the abundance of insects depending on the habitat and the stages of decomposition (p ≤ 0.05; Table 3).

Fourteen dipteran species attracted to rat carcasses in the botanical garden were identified as the following: Chrysomya albiceps (Wiedemann, 1830), Chrysomy megacephala (Fabricius, 1794), Musca domestica (Linnaeus, 1758), Musca sorbens (Wiedemann, 1830), Atherigona sp., Sarcophaga dux (Thomson 1868), Sarcophaga ruficornis (Fabricius, 1794), Megaselia scalaris (Loew, 1866), Drosophila birchii (Dobzhansky & Mather, 1961), Drosophila jambuli (Parshad & Paika, 1965) (D. jambuli), Leucophenga sp., Physiphora alceae (Preyssler, 1791), Chiastocheta trollii (Zetterstedt, 1845) and Crossopalpus aeneus (Walker, 1871). There were also many flies that were identified only to the family level, belonging to the families Milichiidae, Ephydridae, and Psychodidae. On other hand, rat carcasses placed on the building rooftop attracted only nine dipteran species (C. albiceps C. megacephala, M. domestica, M. sorbens, S. dux, Me. scalaris, Leucophenga sp., P. alceae, and Atherigona sp. Table 4). Nine dipteran species (C. albiceps C. megacephala, M. domestica, M. sorbens, Atherigona sp., S. dux, M. scalaris, P. alceae, and Ch. trollii) were attracted to mice carcasses placed in the botanical garden, with only four such species attracted to those placed on the building roof (Table 4).

From the previous results, it is clear that greater number of species were attracted to rat carcasses than mice, placed in the same location. Also more insect species were attracted to carcasses in the botanical garden than on the building roof. Also, results showed that a greater number of species are attracted to rat carcasses than mice carcasses placed in the same location. Additionally, regardless of the rodent type, more insect species were attracted to carcasses in the botanical garden than on the building roof (Fig. 3).

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Fig. 3

Abundance of flies in the two different study habitats, botanical garden and rooftop.

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In addition to species number, the total number of flies was also affected by carcass type and location. Tables 3–6 showed the mean fly abundance and the number of flies attracted to different decomposition stages of rat and mice carcasses in different habitats.

For rat carcasses placed in the botanical garden, C. albiceps and M. domestica were the two most abundant species, followed by M, sorbens, Atherigona sp, S. dux, and P. alceae. For rat carcasses placed on the roof of the building, M. domestica was the most abundant species by far, followed by C. albiceps, M. sorbens, and S. dux. In comparison, for mice carcasses, C. albiceps was the most abundant species in the botanical garden, followed by S. dux and Atherigona sp., while C. albiceps was also the dominant species on the building roof.

This study indicates that there are clear relationships between specific fly species and stages of decomposition (Table 6). The fresh stage attracted only four species of fly and a low number of specimens in general. Drosophila birchii was the most abundant species at this stage. Contrastingly, at the bloat stage, nine species were attracted; C. albiceps was the most abundant species, followed by M. domestica and M. sorbens. All fly species recorded in this study were observed at the decay stage with the exception of D. birchii and D. jambulina. Chrysomya albiceps flies were the most frequently recorded overall during this stage (p ≤ 0.05). At the dry stage, 12 fly species were frequently recorded at low numbers; M. domestica was the most abundant species at this stage.