Carcass placement

Water deer (H. inermis) carcasses, weighing 12 kg, were used. Carcasses were donated by the Chungnam Wildlife Rescue Center. Frozen carcasses were collected and tested. A hill in Jangsu-gun, Jeollabuk-do was selected as the study site (Fig. 1). The summer experiment was conducted twice, in August 2021 and July 2022, whereas the winter experiment was conducted twice, in December 2021 and December 2022. The two carcasses were installed in two places at the experiment location in Jangsu-gun, Jeollabuk-do (35° 34’ 53.41” 127° 25’ 21.76”). To prevent damage to the surrounding wild animals, a 180×100×100 cm rectangular cage was constructed and installed, as shown in Fig. 2.

Observation of decomposition and insect identification

The decomposition stages of the exposed carcasses were divided into fresh, bloated, active, advanced, and remaining (Centeno et al., 2002). For climate observations, the temperature and humidity at the site were directly measured using a digital hygrothermometer (T025, CAS, Seongnam, Korea), and precipitation was recorded using the automatic weather station of the Korea Meteorological Administration. A Timeraps Trail Cam (HP2X; Reconyx, Holmen, WI, USA) was installed to observe the carcasses, and photographs were taken at hourly intervals to monitor the decomposition process. We mainly collected species suspected to be necrophagous, such as insects breeding directly inside or feeding on carcasses. The adults, pupae, and larvae were collected to avoid spiders and other non-insect arthropods. After collection using an insect net, forceps, and spoon, the samples were stored in 50 mL conical tubes containing 75% ethanol. The collected insects were identified by referring to various illustrated books and literature (Buck et al., 2009; Falk, 2016; Ji et al., 2021; Park & Moon, 2020; Szpila, 2009), and genetic analysis was performed using the cytochrome oxidase subunit I primer PolyLCO 5’-GAYTATWTTCAACAAATCATAAAGATATTGG-3,’ PolyHCO 5’-TAMACTTCWGGGTGACCAAARAATCA-3’ (Carr et al., 2011).

Carcass decomposition and insect fauna in the summer and winter of 2021

The highest recorded temperature in the summer of 2021 was 28°C, while the lowest was 18°C. The average humidity was 64% and approximately 30 days were required for the complete decomposition of the carcasses. In the winter of 2021, the highest temperature was 14°C, the lowest temperature was –11°C, and the average humidity was 35%. Complete carcass decomposition took approximately 120 days (Fig. 3).

In summer, the carcass attracts many blowflies and other insects because of the high temperature immediately after installation. As decomposition progressed, additional insects, such as burying beetles, were observed. As the hot and humid environment persisted, the carcass decomposition progressed rapidly (Fig. 4). Immediately after the carcasses were placed, Calliphoridae insects, such as Chrysomya pinguis, were attracted quickly. They accumulate mainly in the eye and mucous membranes. After approximately 5 days, decomposition progressed significantly, and Ptomascopus morio was found to breed inside the body.

Creophilus maxillosus and Vespa velutina nigrithorax were identified and were found to mainly hunt adult and larval flies (Fig. 5). After approximately 1 week, the drying of the carcass was delayed owing to precipitation during the rainy season, with the insides of the carcass turning into sludge. In the advanced decay stage, Necrobia rufipes and Dermestes tessellatocollis were identified on the surface of dry carcass skin, and they were mainly active on the surface of the carcass rather than inside the carcass. These species prefer the dried parts of the carcass and were identified after the carcasses progressed to advanced decomposition. Five orders, 20 families, and 32 insect species were identified during the decomposition (Fig. 6).

In the winter carcass experiment, the temperature remained below freezing, and no insects were identified for approximately 60 days. As in the case studied in Japan in the past (Matoba & Terazawa, 2008), the carcasses did not decompose and remained in their original form, as the activities of microorganisms and insects related to carcass decomposition were significantly reduced because of the environment in which the temperature and humidity were very low. In February, Catops spp. larvae were found to breed as the temperature increased. Contrary to a previous report (Smith, 1986) that flies were the first to be attracted to decomposing carcasses, we found that Coleoptera bred first. The part of the body in contact with the ground was the first to decompose, with larvae found there; this is thought to be because the part in contact with the ground may have been maintained at an appropriate temperature compared with the external environment. Compared to the summer carcasses, the hair removed from the carcasses was preserved without decomposition (Fig. 7). In addition, the occurrence of insects decreased because of the influence of low temperatures. The genus Calliphora appeared more frequently in summer than in summer. Calliphora shows a preference for low temperatures, and their frequency of appearance decreased as the temperature increased. Conversely, the genus Lucilia was not identified at low temperatures but was identified starting in March when temperatures increased. Coleoptera did not appear frequently, and were hidden between the fur and skin of the carcasses. Insect activity only progressed when the temperature rose above freezing. Among these insects, Coleoptera, which burrow directly into the ground, have a reproductive advantage over Diptera, which have difficulty remaining active outdoors at cold temperatures. In winter carcasses, insect emergence is delayed by low temperatures. Coleoptera appear earlier than Diptera; therefore, estimating PMI using Coleoptera rather than Diptera would be more effective in winter. Two orders, six families, and eight species of insects were identified in the winter carcasses. The results are shown in Fig. 8.

Carcass decomposition and insect fauna in the summer and winter of 2022

In the summer of 2022, the highest temperature was 32°C, the lowest temperature was 27°C, and the average humidity was 62%, with approximately 15 days required for complete decomposition of the carcasses. In the winter of 2022, the highest temperature was 20°C, the lowest temperature was –13°C, and the average humidity was 33%, with approximately 120 days required for complete decomposition of the carcasses (Fig. 9).

These summer carcasses, similar to those from the summer of 2021, were the first to attract blowflies. Due to heavy rainfall during this season, carcass decomposition progressed rapidly. Buried beetles were identified as the decomposition progressed. As the hot and humid environment persisted, the decomposition progressed rapidly. Immediately after placing the carcasses, blowflies were the first insects to be attracted. The carcasses decomposed rapidly owing to the constant rainfall (Fig. 10). P. morio and Nicrophorus concolor were identified in decomposed corpses. In the summer of 2021, N. rufipes and D. tessellatocollis were found on the surface of dried carcasses in advanced stages of decomposition. A total of five orders, 17 families, and 29 species of insects were identified according to the progress of carcass decomposition; the results are shown in Fig. 11.

After placing the winter carcasses, the temperature remained below freezing, similar to that in the winter of 2021, and no insects were observed for approximately 50 days. Compared to the summer carcasses, the hair lost from the carcasses was preserved (Fig. 12). In February, when the temperature increased, Catops sp. and O. Colon began breeding (Fig. 13). Calliphora lata growth was confirmed as the temperature rose. Two orders, five families, and seven species were identified; the results are presented in Fig. 14.