TECHNICAL NOTE Helmi Mohd Hadi Pritam, 1 B.Sc. and Paul T. Jayaprakash, 2 Ph.D. Nocturnal Oviposition Behavior of Necrophagous Dipterans in Kelantan, Malaysia* ABSTRACT: The likelihood of dipteran maggots colonizing a corpse due to nocturnal oviposition can be used to challenge the postmortem interval (PMI) estimated assuming diurnal oviposition. Earlier experiments tested nocturnal oviposition behavior by exposing fresh baits once during a single night. In this pilot study, oviposition behavior was studied using beef baits, which, simulating the decay of the body seen in case situations, decomposed inside cages designed to open and close at scheduled intervals during consecutive night or twilight periods. Freshly hatched maggots from diurnally oviposited eggs emerged in control baits on the third day, while a limited number of maggots attributable to nocturnal or twilight oviposition were observed in experimental baits only on the fifth or sixth day, indicating a categorical delay. These results suggest that such delayed and limited nocturnal oviposition is not forensically significant since the larger maggots deriving from diurnal oviposition would be the ones considered when estimating PMI. KEYWORDS: forensic science, forensic entomology, necrophagous dipterans, twilight and nocturnal oviposition, fly-proof cage, postmortem interval The presence of insects and their life stages when colonizing carrion provide reliable parameters for scientific estimation of the time elapsed since death (1–7). Blowflies are generally diurnal and oviposit during the day (1,3–5), though many factors may influence oviposition behavior (3,5). Variables influencing the ovi- position behavior of dipterans include differences in geographical area (3), species differences, seasonal changes, the asynanthropic or urban nature of the habitat and the stage of tissue decay (5). Even ensuring the uniformity of baits left out in fly cages fails to attract flies equally, suggesting the presence of individual idiosyn- crasies (5). Although diurnal, blowflies often lay eggs in dark areas during daytime and it has sometimes appeared that turning off the light in a lab situation can induce egg laying (3). Findings of specific studies on colonization attributable to nocturnal ovipo- sition are diverse; some support oviposition during the night (5,8,9), some find no evidence for such oviposition (1,2,10–14), and a few suggest the possibility of nocturnal oviposition under specific environmental conditions (3,4). Differences in the method- ology of previous studies, such as including artificial lighting (11), ensuring total darkness (9), and allowing (8) or preventing (11) access to the bait by crawling insects may also influence oviposi- tion behavior during the night time. As murders are not field experiments (1,5), the time of oviposition assumed in a crime sit- uation becomes one of the pivotal points in a trial. The prevailing uncertainty relating to nocturnal oviposition prompts challenges to the estimated postmortem interval (PMI) in two ways. First, if PMI is estimated as usual, considering oviposition to be a daytime activity (1,2,10–14), it can be called into question by suggesting the possibility of nocturnal oviposition, as supported by the work of one group of researchers (5,8,9). Second, if PMI is estimated supposing oviposition to be nocturnal (5,8,9), it can be questioned by citing the lack of evidence for nocturnal oviposition reported by the other group of researchers (1,2,10–14). Either way, the estimated PMI can be argued to extend about 10–12 h earlier or later (5)—a duration sufficient to confuse an investigation, or con- vince the judge or jury of an alibi. As there has been no consen- sus on the nocturnal oviposition behavior of necrophagous flies (15), the debate remains inconclusive (16). Previous controlled experiments investigating dipteran colonization due to nocturnal oviposition used fresh baits such as rat carcasses (5,8,12), mutton (9), chicken (11), beef and pig (12), and illegally killed bear cubs (14) that were subjected to a single exposure during the night excluding the influence of decomposition. Be that as it may, in case situations, decomposition is the singular and unique factor that necessitates the use of insects to provide a ‘‘biological clock’’ (5) that more precisely measures the time since death. Further- more, the time ranges for the nocturnal exposure of the bait in the previous studies (8–12) did not include the twilight period. Astronomical twilight occurs near dawn and dusk, and it is defined as the time when the center of the sun is 12–18° below the horizon. Before the beginning of the astronomical twilight in the morning and after the end of the astronomical twilight in the evening, the sun does not contribute to sky illumination (17). Such intermediate levels of illumination during the twilight may affect oviposition behavior differently. In Malaysia, forensically important entomological specimens recovered from human cadavers have been analyzed retrospectively (18–22), and a key was published that identifies prevalent larval species (23). This pilot study was designed to gather empirical evidence on dipteran colonization attrib- utable to twilight and nocturnal oviposition in Kelantan, Malaysia. The same bait was kept in fly-proof cages and exposed during 1 School of Life Sciences, Dundee, DD1 4HN, Scotland, UK. 2 Forensic Science Program, School of Health Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, Malaysia. *Universiti Sains Malaysia is acknowledged for the financial support through Incentive Grant and RU Grant (1001 ⁄ PPSK ⁄ 813011). Received 15 Feb. 2008; and in revised form 21 Aug. 2008; accepted 18 Oct. 2008. J Forensic Sci, 2009 doi: 10.1111/j.1556-4029.2009.01095.x Available online at: www.blackwell-synergy.com Ó 2009 American Academy of Forensic Sciences 1