Molecular Identification Of Forensically Important Diptera Based On Different Length Fragments Of COâ… &COâ…¡ Genes

1Seasonal succession studies for identification of forensically important arthropodsBackgroundForensic entomology, the study of insects pertaining to legal investigations, is primarily concerned with estimations of post mortem interval (PMI). Insect colonization of carrion has been demonstrated to occur in a predictable sequence. There are few available Egyptian studies on the faunal succession related to stages of decomposition and their application in legal medicine. However, there are no published data on the forensically important insects in northeastern Egypt.Objectives1. To identify forensically important species in northeastern Egypt (32°15’E and30°36’N) for the first time.2. To document the arthropod succession pattern (record their presentation at different stages of decomposition).3. To assess rate of decomposition in the2studied seasons.4. To document the early arriving of Diptera (time of arriving and first colonizer).5. To identify the dominant arthropods in each season.Methods1. The2seasonal experiments had been conducted in2consecutive years (2011/2012) by using exposed animal carcasses.2. At each visit, examination of carcasses had been performed to assess the stage of decomposition. Representative samples (dead and still alive) of different life stages have been collected. Environmental variables such as ambient temperature and relative humidity were recorded during each sampling.3. Morphological identification has been conducted by taxonomy experts with using local published keys.Results1. Carcasses yielded3-4orders representing7-9families with11-12collected species in summer and winter experiments respectively. Interestingly, although more species have been collected in winter study but the abundance of each species was much more in summer study.2. To analyze succession patterns of collected species, occurrence matrices were constructed in the summer and winter studies.3. As expected, the rate of decomposition in winter was significantly slower than in summer study.4. There was significance delay in early arriving of Diptera in winter than in summer. The initial colonizers in summer were Musca domestica and Sarcophaga argyrostoma while the initial colonizer in winter was Chrysomya albiceps.5. In general, Muscidae and Fanniidae were the dominant in summer study and Sarcophagidae was the dominant in winter.ConclusionThe results of present study affirms that local surveys are critical in identifying what species are present in various areas, and therefore, what species can be expected to be most important in a medico-legal case. It also constitutes a mandatory and primary step to assess the utility of any molecular markers in identification. 2Cytochrome oxidase I gene:utility of short versus long fragments in identification of forensically significant DipteraBackgroundCorrect identification is an important issue from a forensic perspective because flawed forensic evidence may lead to miscarriages of justice. Molecular based method is considered as an important tool in identification toolkit guided with accurate evaluation of the chosen genetic marker. This is the first time to assess and provide molecular data of forensically important species from Egypt either by using short "272-bp"or long "1173-bp" fragment of the mitochondrial cytochrome oxidase I (COI) gene. Moreover, it is considered also the first time to assess and provide data about long fragment of COI from China because up to our knowledge up to date all available studies from china extracted from short fragment. Thus, assessment will be valuable to determine the suitability of these fragments. The collection of some species from2geographical regions is important to evaluate the dual role of these markers in identification of species and population.Objectives-To determine the power of short and long COI fragment in discrimination of Diptera of forensic relevance then compare between2investigated fragments by assessing:1. Intra and interspecific variations.2. Monophyletic separation of investigated species.3. NJ tree has been constructed consistent with taxonomic classification.-To determine the ability of these fragment to discriminate within same species on population level. Methods1. Morphological identification (50specimens belonging to18species).2. DNA extraction was performed for all examined specimens.3. The2investigated markers were amplified by polymerase chain reaction (PCR) followed by direct sequencing.4. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.Results1. Both tested fragment displayed overlapping between intra and interspecific variations.2. Long marker outperforms short one in completeness of monophyletic separation with high bootstrap support.3. NJ tree based on long fragment clustered species more in accordance with their taxonomic classification than in short fragment.Conclusion1. Identification based on long COI fragment is more reliable and safer because it has the ability to optimize divergence particularly for species characterized by low levels of divergence.2. The other advantage of using long fragment is obtaining phylogenetic data that in accordance with that based on morphological taxonomy. That will help in correct identification even new cryptic species when local databases had been constructed.3. COI gene displayed inability to discriminate within same species on population level. 3Cytochrome oxidase Ⅱ gene:identification of forensically significant Diptera based on short fragmentBackgroundAlthough it has been reported that cytochrome oxidase Ⅱ (COII) gene is useful in evolution studies, population genetics and systematics due to the relatively high degree of variation in the region, but there are few publications about identification of forensically relevant flies based on COII alone. Before introducing any genetic marker to practice on real cases, we need to assess that marker. In order to be useful as a forensic tool, it must contain enough variation to generate unique identifiers. Because of the value of short DNA fragment as being simple and cheap as well as promising result of recent study which assessed189-bp COII in discrimination between3Sarcophagidae species in China. There is no data up to date about that fragment from Egypt. Based on these circumstances, the present study assessed the utility of189-bp COII fragment in identification of14species; belonging to Calliphoridae, Sarcophagidae and Muscidae, originating from2different geographical areas.ObjectivesTo evaluate the utility of too small fragment (<200-bp in length) by assessmg:1. Intra and interspecific variations.2. Monophyletic separation of investigated species and correct assignment on family level.Methods1. Morphological identification (53specimens belonging to14species).2. DNA extraction was performed for all examined specimens.3. The investigated marker was amplified by polymerase chain reaction (PCR) followed by direct sequencing.4. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.Results1. No intraspecific variation within same species (0%), while interspecific variation ranged from2%to25%.2. All specimens were properly assigned into correct species and families apart from H. melanura and M. autumnalis.ConclusionAlthough molecular methods are very useful to identify forensically important insect, we propose to use this fragment (too small fragment<200-bp) only in addition to the conventional methods. 4Applicability of PCR-RFLP in identification of forensically important Sarcophagidae (Diptera) based on COI and COII genesBackgroundThe use of Sarcophagidae (Diptera) for PMI estimation is limited since morphological determination is often hampered due to similar characteristics in the larval, pupal and even adult stage. Almost all available PCR-RFLP data extracted from Calliphoridae. Furthermore, there is a debate about the usefulness of PCR-RFLP technique in identification. Therefore, we aimed to assess the utility PCR-RFLP; as simple and inexpensive technique, based on partial COI-COII genes in identification of4common forensically important Sarcophagidae (Flesh flies) in Egypt and China.Objectives1. To assess the utility of partial COI-COII genes; based on direct sequencing, in identification of4common forensically important Sarcophagidae (Flesh flies).2. To assess the utility of PCR-RFLP; as simple, fast and inexpensive technique based on partial COI-COII genes in identification of4common forensically important Sarcophagidae.Methods1. Morphological identification of4species belong to Sarcophagidae.2. DNA extraction was performed for all examined specimens.3. The investigated marker was amplified by polymerase chain reaction (PCR) followed by direct sequencing.4. Both restriction enzymes Hinfl and MfeI has been used on amplified PCR products then electrophoresis to determine the banding profile.5. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.Results1. All examined specimens were assigned to the correct species.2. Combinations of the restriction enzymes Hinfl and MfeI provide4different restriction fragment length polymorphism (RFLP) profiles even among3sympatric species belong to Sarcophaga genus.ConclusionThis study demonstrates that PCR-RFLP based on partial mt COI-COII genes was accurate to identify forensically important flesh fly species.