Tropical Biomedicine 37(2): 333–356 (2020)

Development patterns of necrophagous infesting rabbit carcasses decomposing in Mount Kapur Cave and its surrounding primary forest in Kuching, Sarawak

Nordin, N.H.1, Ahmad, U.K.1, Abdul Rahim, N.A.2, Kamaluddin, M.R.3, Ismail, D.4, Muda, N.W.1, Abdul Wahab, R.1,5 and Mahat, N.A.1,5,6,7* 1Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia 2Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, Kota Samarahan 94300, Sarawak, Malaysia 3Human and Societal Well-being Research Centre, Faculty of Social Sciences and Humanities, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 4Forensic Science Programme, School of Health Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia 5Enzyme Technology and Green Synthesis Research Group, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia 6Centre for Sustainable Nanomaterials, Ibnu Sina Institute for Scientific and Industrial Research, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia 7Centre of Research for Fiqh Forensics and Judiciary, Universiti Sains Islam Malaysia, 71800 Nilai, Negeri Sembilan, Malaysia *Corresponding author e-mail: [email protected] Received 9 September 2019; received in revised form 10 February 2020; accepted 13 February 2020

Abstract. In addition to the scarcity of baseline data on oviposition of necrophagous and completion of their life cycles in the Borneo region, similar data derived from caves remain unreported. Since entomological baseline data can differ from one biogeoclimatic region to another, the lack of such data would limit the practical values of applying entomological evidence in estimating minimum postmortem interval (mPMI). Therefore, this present research that investigated oviposition and completion of life cycles of necrophagous flies infesting rabbit carcasses decomposing in Mount Kapur Cave and its surrounding forest habitat in Kuching, Sarawak merits forensic consideration. In general, 13 taxa of necrophagous flies were identified viz. Hypopygiopsis violacea, Hypopygiopsis fumipennis, Hemipyrellia ligurriens, Hemipyrellia tagaliana, megacephala, , , Chrysomya chani, Chrysomya pinguis, Chrysomya nigripes, Ophyra spinigera and Ophyra chalcogaster, as well as unidentified Sarcophagidae. In addition, Hyp. violacea and Hyp. fumipennis were the two earlier necrophagous flies that oviposited in all rabbit carcasses decomposing in both habitats. While all these necrophagous flies were observed infesting carcasses in Mount Kapur Cave, Hem. ligurriens and Hem. tagaliana were not found infesting carcasses in the surrounding forest habitat. Complete life cycles for six and five different necrophagous species were successfully observed in Mount Kapur Cave and its surrounding forest habitat, respectively. Significant delay in oviposition, as well as longer durations for completing the life cycles in several necrophagous fly species were observed in Mount Kapur Cave when compared with those of surrounding forest habitat (p < 0.05). These findings deserve consideration as the first ever forensic empirical baseline data on oviposition and completion of life cycles for necrophagous flies in Sarawak as well as in a cave habitat, in view of its practical values for estimating mPMI for forensic practical caseworks.

333 INTRODUCTION life cycle, especially in countries with defined seasonal variations such as Forensic entomology deals with summer and winter (Sharanowski et al., evidence for investigating homicide, suicide 2008). Considering that ambient temperature and suspicious deaths, as well as urban and remains largely similar throughout the stored-products for the purpose of law (Rivers year in tropical countries like Malaysia, and Dahlem, 2014). Insect evidence may also the influence of temperature on insects’ prove useful in investigating cases of human development may not be as huge as in negligence and welfare (Gennard, temperate countries; other factors such as 2012). In addition to its major contribution in rainfall (Mahat et al., 2009) may play a the estimation of minimum postmortem bigger role in this forensic context. interval (mPMI), forensic entomological In Malaysia, application of entomological evidence can also be used to answer forensic evidence is rapidly gaining popularity for questions such as the possible cause of death, forensic practice (Syamsa et al., 2015). relocation of human body and secondary Despite the substantial efforts put forth in disposal, as well as human identification forensic entomological studies within (Gennard, 2012; Mahat and Jayaprakash, Peninsular Malaysia, it is evident that such 2013). Pertinently, beyond 72 hours of death, efforts remain geographically imbalanced pathological changes (e.g. algor mortis and with limited data originating from East rigor mortis) might not be able to assist Malaysia (Sarawak and Sabah) (Mahat and forensic pathologists in determining time of Jayaprakash, 2013). Factors necessitating death due to the advanced stages of forensic entomological studies in Sarawak decomposition (Gennard, 2012). In such and Sabah include the fact that they are (a) situation, forensic entomology may be the geographically separated by the South China only available means for proving or Sea, (b) consisted of a larger land area than disproving one’s alibi (Denis et al., 2018). that of Peninsular Malaysia and (c) perceived While estimation of mPMI can be done as more diverse ecologically in its fauna by assessing the growth pattern of the oldest (Mahat and Jayaprakash, 2013). In view of necrophagous insects (e.g. the thermal the indication made by Anderson (2010) that summation approach), interpretation of the data generated in one biogeoclimatic zone insect succession and composition data may should not be regarded as the same for a be more appropriate for bodies recovered different region, specific study focusing on weeks or months after death (Rivers and developing empirical baseline data for Dahlem, 2014). In this context, it is pertinent Sarawak acquires forensic significance. to indicate that the interpretation of Furthermore, because murder is a crime entomological evidence relies largely on punishable by capital punishment (death) in understanding the oviposition and deve- many countries including Malaysia (Penal lopmental patterns of necrophagous insects Code (Act 574), 2015), murder concealment that vary according to variations in acts are generally expected. Murderers may biogeoclimatic factors (Goff, 2009) as well choose secluded locations such as primary as presence of poisons and/or drugs forest, abandoned house or even a cave to (Mahat et al., 2009; Anderson, 2010). Being conceal the victims in their attempts ‘to poikilotherms, necrophagous insects exhibit confuse, hamper, or defeat investigative holometabolous growth with varying or forensic efforts for the purposes of appearances among the different stages of concealing their identity, their connection to development, dependent on the external the crime, or the crime itself” (Turvey 2008). source of heat for maintaining its Interestingly, review of literature does not physiological and biochemical processes reveal any specific study on oviposition (Gennard, 2012; Rivers and Dahlem, 2014). and developmental patterns as well as Therefore, ambient temperature has been successional patterns of necrophagous flies widely reported in the literature as the major on corpses/carcasses decomposing in a cave factor influencing the duration for completing and its surrounding primary forest.

334 Hence, this present study was aimed at daily until the completion of life cycles providing for the first time, the empirical for all necrophagous flies (until day-41 of baseline data on the developmental patterns decomposition). Exclusively for the cave of necrophagous flies infesting rabbit habitat, daily light intensity was measured. carcasses in the two different habitats (viz. cave and the surrounding primary forest) in Decomposition site Kuching, Sarawak, in view of its practical The carcasses were decomposed at two values for estimating mPMI as well as paving different habitats viz. in Mount Kapur Cave the way for scientific application of forensic at Bau, Kuching (1° 22’ 50.88” N, 110° 7’ 10.46” entomology parameters in Borneo region. E, about 70 meters above sea level; altitude.nu, accessed on 24th November 2017) and its surrounding primary forest (1° 22’ MATERIALS AND METHODS 56.44” N, 110° 6’ 57.42” E, about 40 meters above sea level; altitude.nu, accessed on Experimental design 24th November 2017) within the Division of This present research (ACUC approval Kuching, Sarawak. Mount Kapur Cave is a number: USM/IACUC/2017/(109)(877)) limestone cave with clay floor, located at a utilized a total of six freshly slaughtered vector displacement of about 5.7 km female rabbit carcasses (Oryctolagus southwest of the town of Bau, and about 250 cuniculus) (~1.5 kg) for studying the varying meters east of the main entrance of Fairy species of necrophagous insects and Cave in Krokong area (www.google.com.my/ durations for completing their life cycles in maps, accessed on 24th November 2017). The the Mount Kapur Cave and its surrounding study site was an abandoned gold mine, primary forest in Kuching Division of leaving a side chamber occupied by bats Sarawak. Three carcasses were decomposed (National Resources and Environmental in each habitat during 22nd September – 1st Board, personal communication). On the November 2017. Purchased as carcasses other hand, the chosen habitat of primary from a local rabbit meat seller, the rabbits forest of Mount Kapur Cave as defined by the were sacrificed by slaughtering with the front Sarawak Forestry Corporation (personal part of the neck severed partially. To prevent communication) was located at about 100 m exposure towards insects prior to the from the nearest human establishment. The placement at the decomposition site, the vegetation of this primary forest comprised carcasses were transported in separate of Ficus spp., Melastoma spp. and Calamus sealed double plastic bags and placed at spp. with the estimated canopy coverage of respective decomposition sites before 7.00 about 80% along with the sparse undergrowth a.m. Each carcass was placed directly on low shrubs, wild ferns and bamboo (Mohd- the ground and separated by a minimum Azlan, 2005). Three rabbit carcasses were distance of 20 m apart to minimize left to decompose in each habitat, separated interruption of flies from the adjacent by the minimum distance of 20 m apart from colonies (Mahat et al., 2014). For preventing one another. disturbance by scavengers especially small mammals yet permitting free access for Entomological observation and sampling necrophagous insects, a slotted welded wire Field observation was made at every 2-hour mesh cage (45 cm (length) x 42 cm (width) x interval from 7.00 a.m. to 5.00 p.m. until the 60 cm (height)) was used for each carcass. first observation of the second larvae Following the methodology described by of the first colonizing necrophagous fly Mahat et al. (2009; 2014), entomological species. Subsequently the observation was observations (i.e. the decomposition stages, continued at every 4-hour interval until the presence of adult flies, , larvae, pupae, first observation of pupae, twice daily (around empty pupal cases, tenerals, ambient and 9.00 a.m. and 5 p.m.) during the earlier part of carcass surface temperatures, rainfall as the dry remain stage of decomposition (until well as relative humidity) were recorded day-16) and restricted to once daily (around

335 noon) until the completion of the 41 days of (2012), the number of spiracular slits in the observation period. In this study, the peritremes of posterior spiracles was used description of the decomposition process for assigning the larval instar. In deciding if a provided by Kreitlow (2010) (viz. fresh, particular stage of development had been bloated, decay, postdecay and remains achieved, suggestion made by Clarkson et al. stages) was used. The entomological (2004) was used in this present research. observations recorded during every visit Once the prepupal larvae were observed, the included decomposition stages, presence of surrounding soil area (a minimum of one- adult flies, eggs, larvae, pupae, empty pupal meter radius) was examined for the presence cases, tenerals, ambient and carcass surface of pupae. These pupae were collected and temperatures, daily total rainfall and its reared in a rearing cup in situ (similar to the occurrence, as well as percentage relative rearing cups for larvae) containing sufficient humidity. Using a digital thermometer, the amount of soil to bury the pupae until the in-situ data logger, a hygrometer, as well as emergence of tenerals. The number of pupae a rain gauge, temperatures (carcass surface placed in each rearing cup shall not exceed and ambient), percentage relative humidity 15 for minimizing overcrowding. and the daily total rainfall were recorded, respectively. To measure the light intensity Preservation of larvae and tenerals for in the Mount Kapur Cave, a lux meter was taxonomic identification used. For documenting the findings, a digital The larvae were partially resected trans- camera was utilized. versely on their 11th segment and soaked in Considering the possible presence of 10% potassium hydroxide (KOH) overnight varying species of necrophagous insects on for dissolving the larval tissues. The larvae the carcass, representative specimens of then were placed in glacial acetic acid for larvae (n=10) demonstrating variations in 15 minutes to neutralize the previous KOH physical appearances (e.g. hairy and the solution. At this time, the gut and muscle varying sizes of ) from all contents were removed thoroughly using a masses, as well as soil underneath and around minute pin with a wooden applicator stick as the carcass were collected using surgical its handle. The larvae were then dehydrated forceps and/or brushes. Half of the larvae in ascending ethanol solutions ranging from sampled were killed by immersing them in 80%, 90%, 95% and 100% for 30 minutes for hot water (about 80ºC) and preserved in each solution. The larvae were transferred specimen containers containing 80% ethanol into clove oil for overnight (for coloring) and (Adam and Hall, 2003) for taxonomic soaked in xylene for 30 minutes to wash identification and ascertaining the larval away the clove oil prior to mounting it using instar. Following the procedure suggested by Canada Balsam. As for the adult flies, they Mahat et al. (2009), the remaining half of the were killed using ethyl acetate and pinned larvae were reared in plastic rearing cups with entomological pins for taxonomic containing about 3 cm height of soil secured identification. with cotton gauze and rubber bands until the Taxonomic identification of the third emergence of tenerals for confirming the instar larvae was made by observing the species. To minimize water retention in the morphology of the body region and posterior rearing cup, four tiny holes (diameter: about spiracle (Omar, 2002; Sukontason et al., 2004; 2 mm) at the bottom of the rearing cup were Ahmad-Firdaus et al., 2010; Heo et al., 2015), made. These rearing cups were placed at the while the identification of larval was same habitat where the specimens were attempted by observing the number of collected and examined for the emergence spiracular slits in the peritreme of the of tenerals twice daily (around 9 a.m. and 5 posterior spiracles (Gennard, 2012). Upon p.m.). This was to ensure that the larvae, pre- emergence of tenerals, species identification pupae and pupae developed at similar was made using taxonomic keys provided ambient conditions with that of surrounding by Kurahashi et al. (1997) and Nazni et al. the carcasses. As suggested by Gennard (2011).

336 Statistical analysis between the two habitats (Independent Statistical analyses were performed using Sample t-test, p > 0.05). While the minimum the UTM licensed IBM SPSS 20.0 software. and maximum means of ambient temperature The data analyzed included the ambient in the forest habitat were recorded as 26.51 temperature, total daily rainfall and duration ± 1.22°C and 27.40 ± 1.42°C, respectively, taken for the onset of initial oviposition as the same for the cave habitat were observed well as the completion of life cycles among at 26.41 ± 0.99°C and 27.15 ± 0.66°C, the different species of necrophagous flies correspondingly. Moreover, total rainfall infesting rabbit carcasses decomposing in (> 10.0 mm) in the forest habitat was recorded the forest and cave habitats. The Shapiro- on day 3, 11–12, 17, 20, 22, 24, 26, 29, 32, 35 Wilk test was used for examining the and 40 of the decomposition (Figure 1). normality of the data. The data were normally Pertinently, rains throughout the day were distributed whenever the observed p-values recorded on days-11,17 and 40 of decom- for the normality test were greater than position alone, during which the pupation 0.05, and vice versa. While the normally periods for several of forensically important distributed data were further analyzed using necrophagous flies were observed in this independent t-test and ANOVA, the non- present research. As for the percentage parametric Mann-Whitney U test and Kruskal- relative humidity, significant differences Wallis H were utilized whenever the data between the two habitats were not observed violated the assumption of normality. The (Independent Sample t-test, p > 0.05), despite level of significance (α) of 0.05 was used for slightly higher percentages recorded in the inferring any statistical significance. cave habitat. The minimum and maximum means relative humidity for the forest habitat being 75.37 ± 9.78% and 88.67 ± 3.35%, RESULTS AND DISCUSSION respectively; the same for the cave habitat were 80.71 ± 7.11% and 92.22 ± 3.99%, Environmental condition correspondingly (Table 1). The slightly higher Necrophagous flies are poikilotherms mean percentage of relative humidity (Gennard, 2012; Rivers and Dahlem; 2014), observed in the cave habitat can be explained depend largely on environmental heat and by the fact that the water from rain outside hence, their developmental pattern is a factor of that limestone cave dripped along the of ambient temperature (Anderson, 2010; stalactites into the cave, wetting its clay floor. Rivers and Dahlem, 2014). Relative humidity Because of the shaded feature of the cave as (Krietlow, 2010) and rainfall (Mahat et al., well as the type of soil, evaporation of the 2009) have also been reported as extrinsic retained water was observably slower when factors influencing oviposition and deve- compared with that of forest habitat. lopmental patterns of necrophagous flies. Considering general statements that Therefore, in this present research, the some insects are positively phototropic ambient temperature, total daily rainfall (Smith, 1986) ‘while others seek carrion in (outside of the cave) and percentage relative partial or full shade as oviposition sites’ humidity (Table 1, Figures 1 and 2), as well (Rivers and Dahlem, 2014), light intensity as light intensity in the cave (Figure 2) were (lux) was evaluated for the cave habitat. It recorded in situ. As much as the carcass was found that the light intensity in the cave surface temperatures remained similar ranged between 0.75 – 27.40 lux (median: among all the three replicate carcasses 13.40 lux), depending on the angle of light decomposing in each of the habitats, entering the cave. Although the canopy of representative data alone are provided in trees in the forest habitat may provide limited Figures 1 and 2 for forest habitat as well as in amount of shade to the carcasses from direct the Mount Kapur Cave, respectively. sunlight, the habitat was generally bright Results in Table 1 revealed that the throughout the day, and small variations in means of ambient temperature during the 41 sunlight intensity may not impede the days of observation period remained similar behaviour of necrophagous insects. Because

337 338 of extremely limited light intensity in the position. Although it has been advocated that cave habitat, investigating the effect of low pig is the best model to represent human light intensity in the cave habitat on the decomposition (Wang et al., 2017), other infesting necrophagous flies was found like rabbits (Mahat et al., 2009; 2014; pertinent than that of in the forest habitat. 2016) and monkeys (Ahmad and Ahmad, Apart from light intensity, the amount of shade 2009) have also been used. Rabbit carcasses and the mechanical effect of rain, it can be were used in this present research because construed that the carcasses in both habitats of their easy handling during entomological were relatively exposed to similar ambient observation, but large enough to sustain temperature and percentage humidity, as decomposition to support a considerable depicted in Figures 1 and 2. number and diversity of carrion insects (Bharti and Singh, 2003). Its choice was also Decomposition stages supported by previous researchers (Azwandi One of the biggest issues in forensic et al., 2013) that reported insignificant entomological studies is choosing the differences in Shannon-Weiner index (H’), appropriate animal models for decom- Simpson dominance index (C) and Pielou’s

Figure 1. Total daily rainfall (mm) and mean relative humidity (%), as well as means of ambient and representative carcass surface temperatures (°C) during 22nd September – 1st November 2017 in the forest habitat around Mount Kapur Cave, Kuching, Sarawak (1° 22’ 56.44” N, 110° 6’ 57.42” E).

339 Figure 2. Mean relative humidity (%), means of ambient and representative carcass surface temperatures (°C) as well as light intensity (lux) during 22nd September – 1st November 2017 in Mount Kapur Cave, Kuching, Sarawak (1° 22’ 50.88” N, 110° 7’ 10.46” E).

Evenness index (J) for the species com- variations in the number of decomposition positions in rabbit and monkey carcasses. stages, as well as their durations have been Taking into account all these facts, the choice indicated by many previous forensic of utilizing rabbit carcasses made in this entomological studies. While, five stages of present research appears scientifically decomposition for rabbit carcasses were justifiable. reported by in Shenzen, China (Wang et al., It has been acknowledged by many 2017) and El-Qalyubiya, Egypt (Abd El-bar forensic entomologists that decomposition and Sawaby, 2011), only four stages were process is a continuum of events; however, reported in Punjab, India (Bharti and Singh, for providing better understanding on periods 2003). In this regard, Gennard (2012) of insect activities the process has been attributed variations observed in the number divided into several categorical stages of decomposition stages and their durations (Rivers and Dahlem, 2014). In this context, to extrinsic factors such as habitats. Among different insect taxa have been attributed to others, the five categorical stages (viz. fresh, the different stages of decomposition i.e. the bloated, decay, postdecay and skeletal or fundamental aspect for succession-based remains) described by Kreitlow (2010) for approach for estimating PMI. Interestingly, carcasses decomposing on land have been

340 acquiring popularity in tropical countries decomposing in the cave habitat had like Malaysia. Table 2 represents the onset significantly longer decay stage (Table 2, and durations (number of days) of the Mann-Whitney U test, p < 0.05) when decomposition stages observed in this compared with that of forest. present research. While post decay stage of decomposition In this present research, the five stages for carcasses in forest habitat was observed of decomposition, recommended by Kreitlow by about midday on day-5 and lasted until the (2010) were also observed in all female last observation on day-8, the same for cave rabbit carcasses decomposing in both the habitat took place 24 hours later (midday of forest and cave habitats, the details of which day-6) and lasted until end of day-10; the are provided below. The fresh stage of comparison being statistically significant decomposition began from the moment of (Table 2, Mann-Whitney U test, p < 0.05). The death until evidence of bloating was post-decay stage was characterized by the observed, and this stage lasted for 24 hours presence of only bone, cartilage and skin, (Table 2) in both habitats. Subsequently, during which dipterans ceased from being intensification of the catabolism of organic the predominant insects on the carcasses. substrates as well as putrefaction by The carcasses were considered entering the anaerobic bacteria had led to the inflation of remains stage of decomposition when only the abdomen into balloon-like appearance, bones and furs were observed. Considering known as the bloated stage. Due to the that this present study was limited until putrefaction and influence of insect the completion of the life cycle of all activities, the carcass surface temperatures necrophagous flies infesting the rabbit were observed as slightly higher than that carcasses (day-41), the end of the remains of ambient during the bloated stage of stage of decomposition was not observed. decomposition (Figures 1 and 2). During this The remains stage started on day-9 onwards stage, clusters of dipteran eggs were also for carcasses in the forest habitat. On the found. The bloated stage was observed in all other hand, the stage started on day-11 the carcasses in both habitats on day-2 of onwards for carcasses in the cave habitat. It decomposition, and lasted for about 24 hours is pertinent to indicate here that this was the (Table 2). The presence of larval feeding first study reporting about the decomposition masses and bacterial putrefaction had caused stages and their durations for rabbit carcasses deflation of the body of the carcasses, and decomposing in the limestone cave, limiting this signed the onset of decay stage. For suitable comparisons to be made with the rabbit carcasses decomposing in the forest relevant literature. habitat, the decay stage started on day-3 of decomposition, lasted for 48-52 hours and Life cycles of the infesting necrophagous ended by about midday on day-5 of flies decomposition. Although started on the same Since for each habitat all the three rabbit day-3 of decomposition, rabbit carcasses carcasses were decomposed in sites that were nearby, it was observed that the life cycles for all the infesting necrophagous flies Table 2. Day of observation and durations (number of hours) of the individual decomposition stage remained similar, pertained to that habitat. Therefore, the pooled representative data on Stage Forest Cave the life cycles for necrophagous flies in both the cave and forest habitats alone are Fresh Day 1 (24) Day 1 (24) tabulated here (Tables 3-8). It is pertinent Bloated Day 2 (24) Day 2 (24) to indicate here that, despite considerable Decay Day 3-5 (48-52) Day 3-6 (72-76)* Postdecay Day 5-8 (94-96) Day 6-10 (120-126)* numbers of forensic entomologically-driven Remains Day 9 onwards Day 11 onwards studies covering various biogeoclimatic regions in Peninsular Malaysia, specific * Mann-Whitney U test revealed significantly longer (p < 0.05) studies focusing on the Borneo region remain periods of decay and postdecay stages of decomposition in cave habitat than that of the surrounding forest. sparse (Mahat and Jayaprakash, 2013).

341 342 343 344 345 346 347 Review of literature reveals only one specific that of its surrounding forest habitat are study in Borneo reporting the species of adult provided in Tables 9 and 10. Unfortunately, flies of forensic importance from two peat statistical comparison for specimens of swamp areas in Kuching, Sarawak (Maramat Sarcophagidae between Mount Kapur Cave and Abdul-Rahim, 2015). Considering that and its surrounding forest habitats was not species composition and durations for able to be attempted due to inability to completing life cycle for necrophagous taxonomically identify their species. This insects can vary due to differences in is consistent with indications made by biogeoclimatic regions, estimation of PMI previous researchers that identification of based on baseline data derived from one sarcophagid flies as well as their larvae is region for use in another can be erroneous difficult due to limited taxonomic keys (Anderson, 2010). Because similar studies (Gennard, 2012; Rivers and Dahlem, 2014). focusing on cave habitats, as well as primary Furthermore, the complete life cycle for forest in Sarawak are unavailable, this Sarcophagidae in the surrounding forest present research that was conducted in such habitat was unable to be observed. habitats as Mount Kapur Cave and its In this present research, Hyp. violacea surrounding primary forest in Kuching, and Hyp. fumipennis were invariably Sarawak, appears forensically relevant. observed as the first two necrophagous It is pertinent to note that 13 taxa of species that oviposited on rabbit carcasses necrophagous flies were identified viz. decomposing in both the Mount Kapur Cave Hypopygiopsis violacea, Hypopygiopsis and the surrounding primary forest habitats fumipennis, Hemipyrellia ligurriens, in Kuching, Sarawak (Tables 3 and 4). This Hemipyrellia tagaliana, Chrysomya finding is consistent with that reported by megacephala, Chrysomya villeneuvi, previous researchers (Omar et al., 1994a; Chrysomya rufifacies, Chrysomya chani, Chen et al., 2008) on monkey carcasses Chrysomya pinguis, Chrysomya nigripes, decomposing in a rubber plantation and a Ophyra spinigera and Ophyra chalcogaster, tropical rainforest in Peninsular Malaysia. In as well as an unidentified Sarcophagidae this context, quotations that ‘both species were found infesting the carcasses. However, which are considered the least studied and complete lifecycles were only successfully are the only two species recorded in this observed in six (Hypopygiopsis violacea country’ (Silahuddin et al., 2015) and (Macquart), Hypopygiopsis fumipennis ‘Hypopygiopsis as its biology and ecology (Walker), Hemipyrellia ligurriens are largely unknown’ (Heo et al., 2015) (Wiedermann), appear relevant to this present research, (Fabricius), Chrysomya villeneuvi (Patton) justifying for focusing on these two species. and Chrysomya rufifacies (Macquart)) and Because review of literature does not reveal five (with exception of Hem. ligurriens) any field experiments in Malaysia as well as species in Mount Kapur Cave and its its neighboring countries that explore the surrounding forest, respectively (Tables 3-8). durations for completing the stages of life As for other species, they were either found cycles for Hyp. violaceae and Hyp. as isolated larvae, pupae or tenerals, and fumipennis, suitable comparisons with the therefore, specifying their time of arrival on findings reported here cannot be made. The the decomposing rabbits was not possible. only available data pertaining to these two The inability to categorically record the necrophagous species were obtained from completion of lifecycles for them can be laboratory-controlled experiments (Chen et attributable to the geographical conditions al., 2008; Heo et al., 2015). of the cave (dark) and forest (hilly and While oviposition by both Hyp. violaceae covered with vegetation) that rendered and Hyp. fumipennis in the surrounding difficulties in entomological observation. forest habitat was observed by 9 a.m. on the Suitable statistical comparisons between first day of decomposition, the same was the life cycles of necrophagous flies observed statistically delayed (Table 9, p < 0.05) to in Mount Kapur Cave when compared with 7 a.m. to the next day (day-2) in Mount

348 349 350 Kapur Cave (Tables 4 and 5). The delayed such period and/ or the night before that oviposition by these two necrophagous rendered the soil at the habitat soaked with species in the carcasses in Mount Kapur water. This argument is consistent with the Cave can be attributed to the fact that indications made by previous researchers necrophagous flies are diurnal, whereby they that ‘rain-soaked soil delays development’ are more active in habitat with the presence (Greenberg and Kunich, 2002) and ‘rainfall of light sources (Gennard, 2012; Rivers and of about 9.0 mm or more a day during the Dahlem, 2015) than that of dark habitat, as period of pupation that rendered the soil wet well as in the night (Pritam and Jayaprakash, consistently prolonged the period of pupation’ 2009). In this situation, oviposition on (Mahat et al., 2009). carcasses in the Mount Kapur Cave occurred The total duration for completing the life during the bloated stage of decomposition that cycle for Hyp. violaceae observed in this was associated with the presence of intense present research was substantially longer decomposition odor. On the other hand, from that of 308.25 ± 8.25 hours (equivalent oviposition by these two species observed in to 12-13 days) reported by Chen et al. (2008) carcasses decomposing in the surrounding in their laboratory-controlled experiment. In forest habitat occurred during the fresh stage contrast, the duration for completing the life of decomposition, probably attributable to cycle for Hyp. fumipennis in this present their ready availability in the habitat. As for research was shorter than the 62-72 days the first, second and third instar larvae, as reported by Heo et al. (2015) in their well as the postfeeding larvae of Hyp. laboratory. Interestingly, Heo et al. (2015) violaceae, the stages were observed on day- also reported a personal communication with 2 (7 a.m.), day-2 (5 p.m.), day-3 (3 p.m.) and the Institute for Medical Research, Kuala day-4 (9-11 a.m.) in carcasses decomposing Lumpur indicating that the completion of in the surrounding forest habitat, respectively. life cycle for this particular necrophagous As for the carcasses in the Mount Kapur Cave, species in the insectarium being 19-23 the same stages for Hyp. violaceae were days. The variations observed here when observably delayed by about one day than compared with the two laboratory-controlled those in the forest habitat, attributable to experiments can be explained by fluctuation the delayed oviposition observed earlier of the environmental conditions that (Tables 3 and 9). In addition, significantly prevailed at the decomposition sites, as well longer periods of postfeeding larvae-pupae as other extrinsic factors that merit further and pupae-tenerals, as well as the total research. duration for completing the life cycle for In contrast to the findings reported in Hyp. violaceae were observed in the Mount Peninsular Malaysia (Heo et al., 2007; Mahat Kapur Cave (918 hours, 38.25 days) when and Jayaprakash, 2013; Mahat et al., 2009; compared with that of the surrounding forest 2014; 2016, Denis et al., 2018), C. habitat (554-578 hours, 23.08-24.08 days) megacephala was not found as the first and (Tables 3-4 and 9, p < 0.05). dominant necrophagous fly to oviposit in Interestingly, while no significant rabbit carcasses decomposing in Mount difference in the total duration for completing Kapur Cave and the surrounding forest the life cycle for Hyp. fumipennis between habitats. Although oviposition by C. the two habitats was observed (938 hours, megacephala was observed on the first day 39.08 days), significantly longer durations of decomposition in the forest habitat (11 for the emergence of third instar larvae (from a.m.) (Table 5), the event occurred later than the second instar) as well as tenerals (from that of Hyp. violaceae and Hyp. fumipennis. the pupae) were observed in the surrounding Nonetheless, the observation of oviposition forest habitat than that of Mount Kapur by C. megacephala on the first day of Cave (Table 9, p < 0.05). The longer durations decomposition remains consistent with observed during such stages may be previous findings reported in Peninsular attributable to the presence of heavy rains Malaysia (Heo et al., 2007; Mahat and (Figure 1: day-2, 3, 26, 29, 32, 35 and 40) during Jayaprakash, 2013; Mahat et al., 2009; 2014;

351 2016; Denis et al., 2018). The same pattern rufifacies was observed during the third instar of delayed oviposition for Hyp. violaceae larval stage alone. Such behavior has also and Hyp. fumipennis observed in the been reported by previous researchers in Mount Kapur Cave was also evident for C. Peninsular Malaysia (Omar et al., 1994b; megacephala. Oviposition of C. megacephala Mahat and Jayaprakash, 2013). These in the Mount Kapur Cave was observed authors indicated about the predatory between 11 a.m. to 1 p.m. on day-2 of behavior of the third instar larvae of C. decomposition i.e. at median of 30 hours villeneuvi and C. rufifacies, attributing the when compared with only 4 hours in the behavior to their larger larval size than the forest habitat (Table 9, p < 0.05). The earliest prey. In this context, (a) the predatory observations of the first, second and third behavior of the second instar larvae of C. instar larvae of C. megacephala in the villeneuvi, as well as (b) the ability of the surrounding forest habitat were recorded on smaller larvae viz. C. villeneuvi and C. day-1 (5 p.m.), day-2 (7-9 a.m.) and day-3 rufifacies to predate larger larvae e.g. (3-5 p.m.), respectively (Table 5). In contrast, Hypopygiopsis species observed here have the same stages of larvae were seen on never been reported so far. Because of this day-3 (7 a.m.), day-3 (3-5 p.m.) and day-4 behavior, other larvae of necrophagous flies (7-9 a.m.), correspondingly in Mount Kapur were observably moved away from the Cave (Table 4). Significantly longer periods maggot masses of these two predatory of first-second instar (15.33 ± 1.15 hours) and species on the carcasses. second-third instar larvae (32 (30-32) hours) While the first, second and third instar of C. megacephala were observed in the larvae, as well as postfeeding larvae, pupae surrounding forest habitat when compared and tenerals for C. villeneuvi in the with that in Mount Kapur Cave (Table 9, p < surrounding forest habitat were observed 0.05). The prolongation of these stages may on day-2 (7-9 a.m.), day-2 (5 p.m.), day-3 be due to the rain during the night before (7-9 a.m.), day-8 (9 a.m.), day-9 (9 a.m.) and (7.6 mm), as well as heavy rain during the day-13 (9 a.m. – 5 p.m.), correspondingly, the day of observation (11.8 mm, afternoon until same for the Mount Kapur Cave were noted the next morning) (Figure 1). The post feeding on day-3 (7-9 a.m.), day-4 (11 a.m.), day-5 (11 larvae and pupae of C. megacephala were a.m. – 3 p.m.), day-8 (9 a.m. – 5 p.m.), day-9 (9 observed on day-5 (5 p.m.) and day-11 (11 a.m. – 5 p.m.) and day-15 (9 a.m. – 5 p.m.), a.m.) in the forest habitat, respectively. The respectively (Table 6). Significantly longer same were found on day-6 (11 a.m. – 3 p.m.) periods of eggs-first instar, first-second and day-12 (9 a.m.) in the Mount Kapur Cave, instar, second-third instar larvae, as well correspondingly. Apart from the first-second as postfeeding larvae-pupae, and pupae- and second-third larval stages, other stages tenerals of C. villeneuvi that infested of life cycle for C. megacephala did not exhibit carcasses in the Mount Kapur Cave were statistical differences between the two observed when compared with those in the habitats (Table 8, p > 0.05). Similarly, surrounding forest habitat (Table 6, p < 0.05). statistical difference also did not prevail However, significantly longer period of third for the total duration (240-260 hours, about instar-postfeeding larvae of this species was 10-11 days) for completing the life cycle found in the surrounding forest than that between the two habitats (Table 9, p > 0.05). of Mount Kapur Cave (Table 10, p < 0.05). The durations fell well within the reported Prolongation in developmental stages of durations for C. megacephala reported from C. villeneuvi observed in the Mount Kapur field experiments in Peninsular Malaysia Cave can probably be associated with the (Mahat et al., 2009; 2014; 2016; Denis et al., combinatory effect of slightly lower ambient 2018). temperature, substantially lower light In this present research, the predatory intensity and relatively higher percentage and cannibalistic behavior of C. villeneuvi humidity in the habitat versus that of the was notably evident during the second and surrounding forest habitat. The fact that such third instar larval stages; the same for C. combinatory effect on the growth pattern of

352 necrophagous insects has never been p < 0.05). The prolongation in the periods reported in the literature, undertaking further observed here can be attributable to the same endeavor to elucidate this aspect may prove reason as discussed for C. villeneuvi. The to be an interesting forensically related study. total duration for completing life cycle in The significantly longer period of third instar- Mount Kapur Cave (median: 298.0, range: post feeding larvae of C. villeneuvi in the 296.0-298.0 hours, equivalent to 12.4 days) surrounding forest habitat can be explained was significantly longer when compared with by the fact that the soil at that decomposition that of the surrounding forest habitat (median: site was consistently soaked with water 266.0, range: 264.0-266.0 hours, equivalent during the second and third day of to 11.1 days). It appears that the total duration decomposition, attributable to the presence for completing the life cycle for C. rufifacies of rain (Figure 1). While field studies (Omar in the surrounding habitat was consistent with et al., 1994a; 1994b; Silahuddin et al., 2015) the findings reported by Mahat et al. (2016) that reported about C. villeneuvi in in Kubang Kerian, Kelantan. As for the cave Peninsular Malaysia do not explicitly tabulate habitat, suitable comparison could not be its complete life cycle, Mohd Salleh et al. made due to unavailability of the information (2014) attempted to provide such data via in the body of literature. their laboratory-controlled experiment. They For Hem. ligurriens, the first, second reported that C. villeneuvi required 9.00 ± and third instar larvae, as well as postfeeding 0.07, 9.34 ± 0.04 and 9.40 ± 0.02 days to larvae, pupae and tenerals in Mount Kapur complete the life cycle when reared at Cave were observed on day-2 (5 p.m.), day-3 constant temperatures of 30, 27 and 25°C, (7 a.m.), day-3 (5 p.m.), day-5 (1 p.m.), day-7 respectively, with percentage humidity (9 a.m.) and day-23 (11 a.m.), respectively ranged between 70-85%. As for this present (Table 8). Considering that this necrophagous research, the total durations for completing species was not found in the surrounding the life cycle in Mount Kapur Cave (310.00 ± forest habitat, statistical comparison of the 3.46 hours, about 13 days) and its surrounding life cycle between the two habitats cannot forest habitat (290.67 ± 12.20 hours, about be made. Despite being indicated as one of 12 days) were statistically insignificant the important necrophagous flies in forensic (Table 10, p > 0.05). In this context, the longer entomology, review of literature reveals no durations for completing the life cycle for comprehensive data pertaining to the C. villeneuvi observed in this present complete life cycle of Hem. ligurriens in research may largely be due to fluctuations Malaysia. The only available studies (Omar in the environmental conditions (especially et al., 1994a; Silahuddin et al., 2015) were ambient temperature as well as rain and of field observations without explicitly humidity) at the decomposition sites. providing the complete life cycles. In this As for C. rufifacies, its first, second and regard Yang et al. (2015) reported a third instar larvae, as well as postfeeding pioneering laboratory research that tabulated larvae, pupae and tenerals in the surrounding the complete life cycle of Hem. ligurriens forest habitat were observed on day-2 (9 a.m.), at controlled temperatures. It can be made day-2 (5 p.m.), day-3 (7-9 a.m.), day-4 (1-3 up from their Table 1 that Hem. ligurriens p.m.), day-8 (5 p.m.) and day-12 (9 a.m.), required 566.9 hours (equivalent to 23.62 respectively (Table 7). The same for Mount days) to complete its life cycle at the Kapur Cave were: day-3 (9 a.m.), day-3 (5 constant temperature of 28°C and relative p.m.), day-4 (7-9 a.m.), day-5 (3-5 p.m.), day- humidity of 70%. In this present research the 9 (5 p.m.) and day-13 (5 p.m.), correspondingly completion of life cycle for Hem. ligurriens (Table 7). The result revealed significantly in Mount Kapur Cave occurred at 532.0 hours longer periods of third instar-postfeeding (equivalent to 22.2 days), about a day shorter larvae and pupae-tenerals for C. rufifacies than that of Yang et al. (2015). Such a shorter in Mount Kapur Cave when compared with total duration observed here, when compared that of surrounding forest habitat (Table 10, with that reported by Yang et al. (2015), can

353 be attributed to the fact that (a) the LIMITATION decomposition took place in a cave habitat Although morphological differences in aspect with limited amount of light intensity, as ratios, presence of fur, as well as weight well as (b) fluctuation and relatively lower would limit suitable discussions of the ambient temperatures (min: 26.41 ± 0.99! findings for human forensic, rabbit carcasses and max: 27.15 ± 0.66!) at the decom- were used due to its easy handling, as well as position site than that reported by Yang et having sufficient body size to support insect al. (2015). infestation (Bharti & Singh, 2003). Therefore, It has to be acknowledged here that, replicating the same experiment using pigs besides the species discussed above, other that are similar to human bodies would be necrophagous species viz. Hemipyrellia necessary for providing better insights into tagaliana (Bigot), Chrysomya chani rates of decomposition and patterns of fly Kurahashi, Chrysomya pinguis (Walker), colonization. Chrysomya nigripes Aubertin, Ophyra spinigera Stein and Ophyra chalcogaster Acknowledgements. The authors are (Widermann) were also observed infesting thankful to the Universiti Teknologi Malaysia the carcasses. However, due to logistic for providing the Research University Grants problems as well as their limited quantities (Q.J130000.2526.16H92 and Q.J130000. and dispersive behavior, they were either 2526.12H51) that enabled this present found as larvae, pupae or tenerals alone. research to be conducted. Therefore, proper discussion on their complete life cycles could not be made, necessitating further studies focusing on REFERENCES these necrophagous species at the same decomposition sites. Abd El-Bar, M.M. & Sawaby, R.F. (2011). A preliminary investigation of insect colonization & succession on remains of CONCLUSION rabbits treated with an organophosphate insecticide in El”Qalyubiya Governorate Investigation of the species of necrophagous of Egypt. Forensic Science International flies in both Mount Kapur Cave and its 208: e26-e30. surrounding forest habitat revealed the Adams, Z.J.O. & Hall, M.J.R. ( 2003). Methods infestation by 13 different taxa of necro- for killing & preservation of blowfly phagous flies on the decomposing rabbit larvae, & their effect on post-mortem carcasses viz. four species of Calliphorinae larval length. Forensic Science Inter- (Tribe Luciliini: Hyp. violacea, Hyp. national. 138: 50-61. fumipennis, Hem. ligurriens and Hem. Ahmad, A. & Ahmad, A.H. (2009). A pre- tagaliana), six species of (C. liminary study on the decomposition & megacephala, C. villeneuvi, C. rufifacies, C. dipteran associated with exposed chani, C. pinguis and C. nigripes), two carcasses in an oil palm plantation in species of Azelinae (Tribe Azelini: O. Bandar Baharu, Kedah, Malaysia. spinigera and O. chalcogaster) as well as an Tropical Biomedicine 26: 1-10. unidentified Sarcophagidae. Results revealed Ahmad-Firdaus, Marwi, M.A., Syamsa, R.A., that (a) Hyp. violacea and Hyp. fumipennis Zuha, R.M., Ikhwan, Z. & Omar, B. (2010). were the two earlier necrophagous flies that Research Note Morphological descrip- oviposited in all carcasses in both habitats tions of second & third instar larvae of and (b) significant delay in oviposition, as Hypopygiopsis violacea Macquart well as longer durations for completing the (Diptera: ), a forensically life cycles in several necrophagous fly important fly in Malaysia. Tropical species were observed in Mount Kapur Cave Biomedicine 27(1): 134-137. when compared with those of surrounding forest habitat.

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