The uses of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) in forensic entomology

Chrysomya megacephala (Fabricius,1794) occurs on every continent and is closely associated with carrion and decaying material in human environments.Its abilities to find dead bodies and carry pathogens give it a prominence in human affairs that may involve prosecution or litigation,and therefore forensic entomologists.The identification,geographical distribution and biology of the species are reviewed to provide a background for approaches that four branches of forensic entomology (urban,stored-product,medico-criminal and environmental) might take to investigations involving this fly.

Lucilia sericata(Meigen)and Chrysomya megacephala(Fabricius)(Diptera:Calliphoridae)Development Rate and its Implications for Forensic Entomology

Forensic entomology is basically a science that is based on the scientific study of the invasion and succession pattern of arthropods with their developmental stages of different species found on the decomposed cadavers during legal investigations.The developmental rate of Lucilia sericata and Chrysomya megacephala was studied in beef liver for the correlation of time duration in each phase with the temperature and climate.The obtained data belong to L.sericata at temperatures between 22℃and 26℃(mean‑24℃)and relative humidity 50%±10%and C.megacephala at temperatures between 23℃and 27℃(mean‑25℃)and relative humidity 55%±10%.From the analysis of results,it was observed that in the climatic conditions of the study area,time since death assessment involving L.sericata was found to be with a potential of maximum 10-11 days and C.megacephala with 8-9 days.The data emerged as results from the present work would be beneficial for investigations involving decomposed dead body remains for the assessment of time since death.

A brief review of forensically important flesh flies (Diptera: Sarcophagidae)

Forensic entomology could provide valuable data for the minimum postmortem interval (PMImin) estimation and other relevant information,such as causes and circumstances of death.Some representatives of flesh flies are one of the dominant necrophagous insects during early stages of decomposition,demonstrating unique biological characteristics compared with other necrophagous flies.Moreover,they lead to global health concerns as carriers of various pathogenic micro-organisms,and dominantly result in the traumatic myiasis.Thus,sarcophagid flies are considered important in decomposition processes for PMImin estimation.However,the utility of sarcophagid flies has been seriously hampered by limited ecological,biological and taxonomic knowledge of them.The aim of this paper is to provide a brief review on the species,distribution and biological habit of forensically important sarcophagid flies.In addition,the relation between traumatic myiasis and flesh flies,molecular identification methods and developmental pattern of flesh flies are summarized.

Identification of forensically important blow fly species （Diptera： Calliphoridae） in China by mitochondrial cytochrome oxidase I gene differentiation

Unambiguous and rapid sarcosaphagous insect species identification is an essential requirement for forensic investigations. Although some insect species are difficult to classify morphologically, they can be effectively identified using molecular methods based on similarity with abundant authenticated reference DNA sequences in local databases. However, local databases are still relatively incomplete in China because of the large land area with distinct regional conditions. In this study, 75 forensically important blow flies were collected from 23 locations in 16 Chinese provinces, and a 278-bp segment of the cytochrome oxidase subunit I gene of all specimens was successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all Calliphorid specimens were properly assigned into nine species with relatively strong supporting values, thus indicating that the 278-bp cytochrome oxidase subunit one region is suitable for identification of Calliphorid species. The clear difference between intraspecific threshold and interspecific divergence confirmed the potential of this region for Calliphorid species identification, especially for distinguishing between morphologically similar species. Intraspecific geographic variations were observed in Lucilia sericata （Meigen, 1826） and Lucilia caesar （Linnaeus, 1758）.

Chrysomya chani Kurahashi (Diptera: Calliphoridae), a blow fly species of forensic importance: morphological characters of the third larval instar and a case report from Thailand

Blow flies are worldwide the most important insects from a forensic point of view.In Thailand,aside from the two most common species,Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart),Chrysomya chani Kurahashi was also found to be of forensic importance.We present a case of a human female cadaver in its bloated stage of decomposition,discovered at Pachangnoi Subdistrict,northern Thailand.Entomological sampling during the autopsy displayed an assemblage of numerous dipteran larvae.Macroscopic observations showed the coexistence of third instar larvae of the three blow flies C.megacephala,Chrysomya villeneuvi Patton,an unknown blow fly species and one muscid,Hydrotaea sp.The minimum post-mortem interval was estimated to be six days,based on the developmental rate of C megacephala.The ID of the unknown larva,which is the focus of this report,was revealed later as C.chani by DNA sequencing,using a 1205 bp of cytochrome c oxidase subunit Ⅰ (COⅠ).The occurrence of C.chani on a human body revealed the need to analyse and describe the morphology of its immature stage,to enable forensic entomologists to identify this fly species in future cases.The morphological examination of the third instar was performed,revealing peculiar characteristics: protuberant tubercles encircling abdominal segments;9-11 lobes on the anterior spiracle;six prominent pairs of tubercles along the peripheral rim of the eighth abdominal segment;a heavily sclerotized complete peritreme of the posterior spiracles.A key to differentiate the third instar of blow flies of forensic importance in Thailand is provided.

Metrological framework for selecting morphological characters to identify species and estimate developmental maturity of forensically significant insect specimens

Accurate age estimates of immature necrophagous insects associated with a human or animal body can provide evidence of how long the body has been dead.These estimates are based on species-specific details of the insects’aging processes,and therefore require accurate species identification and developmental stage estimation.Many professionals who produce or use identified organisms as forensic evidence have little training in taxonomy or metrology,and appreciate the availability of formalized principles and standards for biological identification.Taxonomic identifications are usually most readily and economically made using categorical and qualitative morphological characters,but it may be necessary to use less convenient and potentially more ambiguous characters that are continuous and quantitative if two candidate species are closely related,or if identifying developmental stages within a species.Characters should be selected by criteria such as taxonomic specificity and metrological repeatability and relative error.We propose such a hierarchical framework,critique various measurements of immature insects,and suggest some standard approaches to determine the reliability of organismal identifications and measurements in estimating postmortem intervals.Relevant criteria for good characters include high repeatability(including low scope for ambiguity or parallax effects),pronounced discreteness,and small relative error in measurements.These same principles apply to individuation of unique objects in general.

Molecular identification of the potentially forensically relevant cluster flies Pollenia rudis (Fabricius) and Pollenia vagabunda (Meigen) (Diptera: Polleniidae) — non-recorded species in Algeria

Cluster flies are represented by the genus Pollenia Robineau-Desvoidy,1830 of the family Polleniidae Brauer and Bergenstamm,1889.Their larvae are known to be internal parasites or predators of earthworms.Herein,we report for the first time the occurrence of the cluster flies Pollenia rudis Fabricius,1794 and Pollenia vagabunda(Meigen,1826)(Diptera:Polleniidae)on carcasses in Algeria and identify them through DNA barcoding.A region of the mitochondrial cytochrome c oxidase I gene(COI)was amplified and sequenced.Genetic distances were determined.A phylogenetic tree was constructed with the maximum parsimony method using 10000 bootstrap replicates.A total number of 157 adults of P.rudis were collected together with 325 adults of Pollenia vagabunda.The occurrence of Pollenia on animal carcasses does not seem to be correlated with a particular stage of decomposition.All the sequences were correctly identified using the BLASTn tool from the GenBank database and the BOLD identification engine.Intra-and interspecific sequence divergence values were less than 1%and greater than 3%,respectively.COI barcodes obtained from this study were robust enough to identify and distinguish unambiguously between P.rudis and P.vagabunda.In the tree-based analysis,the cluster flies were all assigned to their respective species separately from each other confirming the morphological identification.These results provide DNA barcodes that contribute to the growth of reference databases and allow fast and accurate identification.

Mitochondrial and Nuclear DNA-based Identification of Some Forensically Important Calliphoridae(Diptera)in Luoyang of China

Introduction:Calliphoridae plays a key role in forensic entomology research,which is one of the first insects to decompose animal carcasses.The mitochondrial cytochrome c oxidase subunit I and the ribosomal internal transcribed spacer 2(ITS2)are among the most widely used molecular markers for insect taxonomic characterization.Aim:The aim of the study was to test the suitability of two genetic markers based on conducting the molecular identification of six necrophagous Calliphorid flies.Materials and Methods:Fourteen Calliphoridae flies were collected and classified with traditional morphological characteristics.The DNA of flies was extracted and the fragments of COI and ITS2 were amplified and sequenced.All the sequences were aligned and analyzed by MEGA 7 software for NCBI BLAST,nucleotide composition,intra-and inter-specific divergence calculation,and phylogenetic tree inference successively.Results:The results indicated that COI and ITS2 genes were robust in the identification of Calliphoridae at the species level and ITS2 gene sequence possessed a strong resolution power as it showed higher variation values between Lucilia sericata and Lucilia cuprina,Calliphora vomitoria and Triceratopyga calliphoroides,C.vomitoria andAldrichina grahami,but inferior to COI fbrT.calliphoroides and A.grahami.Conclusions:Our results showed that combination of COI+ITS2 genes yields more accurate identification and diagnoses and better agreement with morphological data than the mitochondrial barcodes alone.As a supplementary method for morphological identification,we advocated for the combination of nuclear and mitochondrial gene approaches to address the taxonomy and phylogeny of forensic relevant flies,especially of closely related species and populations.

Seasonality of insect succession on exposed rabbit carrion in Guangzhou, China

Succession patterns for insect fauna, spanning four seasons, on rabbit carcasses were studied in Guangzhou China from 2006 to 2008. A total of 49 taxa belonging to three orders and 15 families were collected and identified. Four decomposition stages were recognized： fresh, bloated, decay and dry stage. Taxa diversity was higher in bloated and decay stages than fresh and dry stages. Internal and ambient temperatures were measured. Maximum internal temperature arrived earlier in warmer seasons. The sequence of insect succession in this study follows the same general patterns： the Diptera peaked initially, and the Coleoptera peaked later. Chrysomya megacephala was the dominant species in four seasons. Its time point of different development stages on carrion could be the most important indicators for PMI （postmortem interval） estimation in Guangzhou. Yearly and seasonal differences in activities of insects were compared. Alpha diversity indices were calculated. There were no significant differences in Shannon-Wiener index, evenness （J） and dominance （C） between seasons and years. From the Jaccard similarity index, the highest similarity was between winter and spring, while the lowest was between summer and winter. Some species occurring exclusively in one season may be important to characterize different seasons. A list of the insect taxa associated with above-ground rabbit carrion during the four seasons was compiled, which can be used for estimating time since death in similar bio-climate areas.

Development of Chrysomya megacephala at constant temperatures within its colony range in Yangtze River Delta region of China

Chrysomya megacephala (Fabricius,1794) is the most abundant and predominant species which arrives and colonizes a cadaver first in most parts of China.Therefore,its growth and development patterns have great implications in the estimation of the minimum postmortem interval (PMImin).In this study,C megacephala was collected from the Yangtze River Delta region and reared at seven constant temperatures ranging from 16 ℃ to 34 ℃.The developmental duration and accumulated degree hours,larval body length and morphological changes of C.megacephala were examined.Furthermore,we constructed three developmental models,isomorphen diagram,isomegalen diagram and thermal summation model,which can be used for estimating PMImin.The developmental durations of C.megacephala at 16 ℃,19 ℃,22 ℃,25 ℃,28 ℃,31 ℃ and 34 ℃ are (794.8 ± 14.7),(533.2 ± 10.1),(377.8 ± 16.8),(280.8 ± 15.1),(218.9 ± 8.5),(190.8 ± 10.1) and (171.8 ± 6.8) h,respectively.The developmental threshold temperature D0 is (11.41 ± 0.32) ℃,and the thermal summation constant K is (3 418.7 ± 137.0) degree hours.Regression analysis was conducted to obtain equations of the variation in larval body length with time after hatching,and variation in time after hatching with body length.Moreover,our study divides the intra-puparial morphological changes of C.megacephala into 11 sub-stages,and provides the time range experienced by each sub-stage.The results of this study provide fundamental development data for the use of C.megacephala in PMImin estimations.

Effect of low temperature in the development cycle of Lucilia sericata (Meigen) (Diptera, Calliphoridae): implications for the minimum postmortem interval estimation

Knowledge of necrophagous insects' developmental data is necessary for the forensic entomologist to estimate a reliable minimum postmortem interval (PMImin).Among the most represented necrophagous species,Lucilia sericata (Diptera,Calliphoridae) is particularly interesting.It is regularly identified in samples,with a predominance in summer,and is commonly used by analysts of our entomology department (Institut de Recherche Criminelle de la Gendarmerie Nationale) to estimate the PMImin with the accumulated degree days (ADD) method.This method requires the mathematical lower thermal threshold to be known.This value dictates the quality of the applied ADD method but cannot be considered as fixed,especially when insect development occurs at temperatures close to the biological threshold.In such conditions,it is necessary to study the influence of such temperatures on development rate,as well as the consequences of estimating the period of first oviposition on cadavers,when using the ADD method.Seven replicate rearings were conducted at six different temperatures: 30 ℃,24 ℃,18 ℃,15 ℃,12 ℃ and 10 ℃.Time of development and time of emergence were recorded.The effect of low temperature on the development cycle and the reliability of the ADD method under this entire temperature spectrum were studied using different linear regression models.Calculated durations of total insect time development and experimental rearing duration were then compared.A global linear model cannot be used on the whole temperature spectrum experienced by L.sericata without resulting in an overestimation at some temperatures.We found a combination of two linear regression models to be suitable for the estimation of the total development time,depending on the temperature experienced by L.sericata.This approach allowed us to obtain a variation lower than 2% at 12 ℃ and 10 ℃ between the calculated duration and experimental duration of development.In comparison,the results obtained with a global model show a variation higher than 3% at 12 ℃ and 10%at 10℃.

To eat or get heat： Behavioral trade-offs between thermoregulation and feeding in gregarious necrophagous larvae

The thermoregulation behavior ofLucilia sericata larvae （Diptera： Calliphori- dae）, a necrophagous species that feeds on vertebrate cadavers, was investigated. These larvae require high heat incomes to develop, and can elevate temperatures by forming large aggregates. We hypothesized that L. sericata larvae should continue to feed at temperatures up to 38 ℃, which can be reached inside larval masses. Thermal regulation behavior such as movement between a hot food spot and colder areas was also postulated. The hypotheses were tested by tracking for 1 h the activity of single, starved third instar larvae in a Petri dish containing 1 food spot （FS） that was heated to a constant temperature of 25 ℃, 34 ℃ or 38 ℃ with an ambient temperature of 25 ℃. The influence of previous conspecific activity in the food on larval behavior was also tested. The crops of larvae were dissected to monitor food content in the digestive systems. Based on relative crop measurements, larvae fed at all food temperatures, but temperature strongly affected larval behavior and kinematics. The total time spent by larvae in FS and the duration of each stay decreased at high FS temperature. Previous activity of conspecifics in the food slightly increased the time spent by larvae in FS and also decreased the average distance to FS. Therefore, necrophagous L. sericata larvae likely thermoregulate during normal feeding activities by adjusting to local fluctuations in temperature, particularly inside maggot masses. By maintaining a steady internal body temperature, larvae likely reduce their development time.

Benefits of heterospecific aggregation on necromass: in fluence of temperature, group density, and compositi on on fitness-related traits

Necrophagous blowflies(Diptera:Calliphoridae)such as Calliphora vicina,a cold-tolerant species,and L ucilia serie ata,a warm-adapted species,are pioneer carrion・breeder.Although these two species have different temperature preferences,larvae aggregate actively and often feed simultaneously on carrion.The hypothesis to be tested was that L.sericata benefits from the association with C.vicina at lower temperatures(i.e.,15℃)and that C.vicina derives greater benefits from this association at higher temperatures(i.e.,28℃).Therefore,both species were raised at these two constant temperatures from first instars to adults under three different conditions:monospecific low-density,monospecific high-density,and heterospecific high-density.The time until larval migration,surface area of puparia,and survival rates were determined for each condition.Differences between these fitness-related traits were found between species,temperatures,group densities,and species compositions.At 28°C,C.vicina larvae bred in heterospecific groups migrated significantly earlier and in higher numbers than that under same density conspecific con・ditions,with a lower mortality rate.At 15°C,both species benefited from high-density heterospecific associations,expressed by faster development and larger puparia.In conclusion,necrophagous larvae benefited from heterospecific aggregations at suboptima 1 temperatures by adapting their migration time to that of the faster species.Since temperature changes throughout the day and over the year,the beneficiary of such a collective association also changes.The costs involved and deviations to the temperature-size rule highlight the complexity of the carrion ecosystem.

Detection of food source by PCR analysis of the gut contents of Aldrichina grahami （Aldrich） （Diptera： Calliphoridae） during post-feeding period

The last meal of sarcophagous maggots may be useful in identifying the species on whose flesh they have fed （the＂host＂species）. The DNA profile of the host species may indeed be detectable in the＂last meal＂. In this paper, mitochondrial DNA analysis of gut contents was used to identify the prior host of post-feeding larvae of Aldrichina grahami （Aldrich） （Diptera： Calliphoridae）. A modified logistic equation was fitted to estimate the probability of identifying the host under five different constant temperatures （16, 20, 24, 28 and 32 ℃）. Our results shows that the detected time ranged from a maximum of 24 h at 32℃ to 42 h at 16℃ and a minimum of 12 h at 32~C to 30 h at 16℃. Furthermore, the host detection time was also calculated to give the maximal time after larval hatching from the egg. These results indicate that, in criminal cases where the maggots stray from the corpse, the last meal of the larvae should not be overlooked as potentially critical evidence.

Variation in decomposition stages and carrion insect succession in a dry tropical climate and its effect on estimating postmortem interval

Insects have an important role in minimum postmortem interval(PMImin)estimation.An accurate PMImin estimation relies on a comprehensive study of the development and succes-sion of local carrion insects.No published research on carrion insect succession exists for tropical north Queensland.To address this,we aimed to obtain preliminary observational data concerning the rate of decomposition and insect succession on pig carcasses in Townsville and compare these with other regions of Australia and overseas.Adult insects were collected daily from three pig carcasses for 30d during summer and identified to fa-mily level.Observations of decomposition rate were made each day and progression through the stages of decomposition were recorded.Adult insects were identified to family and their presence/absence used as a proxy for arrival at/departure from the remains,respectively.These preliminary data highlight several interesting trends that may be inform-ative for forensic PMImin estimation.Decomposition was rapid:all carcasses were at the dry/remains stage by Day 5,which was substantially quicker than all other regions in the com-parison.Differences were also observed in the presence/absence of insect families and their arrival and departure times.Given the rapid progression through early decomposition,we argue that later-arriving coleopteran taxa may be more forensically informative in tropical Australia,in contrast with temperate regions where Diptera appear most useful.This research contributes preliminary observational data to understanding insect succession pat-terns in tropical Australia and demonstrates the critical need for comprehensive local succes-sion data for each climatic region of Australia to enable accurate PMImin estimation.These data will inform future research targeted at gaining a more comprehensive understanding of insect succession in the Australian tropics.

Histological age estimation of the eggs of Calliphora vicina Robineau Desvoidy (Diptera: Calliphoridae)

Aging blow fly eggs can be critical to a forensic investigation,but there are currently no forensically useful timelines describing internal anatomical changes in embryological development.This is partly due to the lack of an economical,rapid and technically simple histological technique to allow mass production of slides for research and casework.We present a histological method that uses a slightly modified standard laboratory processing run with 1 h fixation in 10% formalin,2 h softening in Molliflex and Haemotoxylin and Eosin (H&E) staining.We also present a summary of the internal anatomical changes that can be visualized using our technique in the developing eggs of Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae).We examined eggs from at least three different females grown at 15 ℃ and sampled at 6 h intervals,and eggs grown at 20 ℃ and sampled at 3 h intervals.Blind aging trials demonstrated that it is possible to accurately age material grown at 20 ℃ to within 6 h (but attempts to further narrow this interval resulted in errors in one-third of cases).It was also possible to see sufficient anatomical detail to age eggs preserved for forensic casework 5,8,9,10 and 11 years previously.Additionally,we determined that section quality was improved by 5 s fixation in hot water prior to preservation in ethanol.However,hot water fixation for longer than this increased the level of section artefact.

Amplified fragment length polymorphism analysis supports the valid separate species status of Lucilia caesar and L.illustris (Diptera: Calliphoridae)

Common DNA-based species determination methods fail to distinguish some blow flies in the forensically and medically important genus Lucilia Robineau-Desvoidy.This is a practical problem,and it has also been interpreted as casting doubt on the validity of some morphologically defined species.An example is Lucilia illustris and L.caesar,which co-occur in Europe whilst only L.illustris has been collected in North America.Reports that these species shared both mitochondrial and nuclear gene sequences,along with claims that diagnostic morphological characters are difficult to interpret,were used to question their separate species status.We report here that amplified fragment length polymorphism profiles strongly support the validity of both species based on both assignment and phylogenetic analysis,and that traditional identification criteria based on male and female genital morphology are more reliable than has been claimed.

Diversity of the Formicidae (Hymenoptera) carrion communities in Lisbon (Portugal): preliminary approach as seasonal and geographic indicators

The value of the Formicidae (Insecta,Hymenoptera) community in forensic investigations is poorly studied in Portugal.In order to better understand the structure and dynamics of this group of insects in cadavers and their putative value in forensic investigations,studies were carried out in Lisbon area during one year.Piglet carcasses were used as a model of human decomposition.The entomofauna attracted to the carcasses was collected over a period of about 2 months in each season of the year.The collection of ants was performed at regular intervals,daily in the first 23 days and then with intervals of 2,3 or 5 days until the end of the experiment.Five stages of cadaveric decomposition were recognized with the ants being present in all of them.Three hundred and nine specimens were captured: 7 in autumn,6 in winter,90 in spring and 206 in summer.These specimens belong to three subfamilies and seven different species.Four of them had never been mentioned before as being associated to cadavers in this geographical area.Spring and summer had the greater diversity of species and a larger number of individuals.Temnothorax luteus was the dominant species in spring and Tapinoma nigerrimum in summer.Corroborating the information of other similar studies carried out in the Iberian Peninsula,the sarcosaprophagous Formicidae community found in Lisbon is unique and different from other studied locations,which supports the need to perform regional studies.Our results shown that ants do not present a definite pattern of succession,but some species have the potential to be seasonal or geographic indicators.

Record of Leptometopa latipes(Diptera:Milichiidae)from a human cadaver in the Mediterranean area

In forensic entomology,insects are used mainly to obtain information about the time since death.This information is obtained studying the developmental rate of the first colonizers,principally species in the families Calliphoridae,Sarcophagidae,Muscidae,Stratiomyidae and Phoridae.However,species belonging to other families can provide information about body transfer or the season of the death.Among them Milichiidae are flies rarely reported from human cases despite the larvae of some species are known as saprophagous feeding on plant and animal decomposing matter.A potential cause of the lack of records of these species from forensic cases can be related with the paucity of descriptions and illustrations of the immature stages.In this article,the entomological samples collected from a human body found inside an apartment in a Maghreb country,in Northern Africa,is reported and Leptometopa latipes(Diptera:Milichiidae)is described in detail.Molecular analysis is also reported to confirm the morphological analysis.

Delayed reception of live blowfly (Calliphora vicina and Chrysomya rufifacies)larval samples: implications for minimum postmortem interval estimates

Forensic entomology evidence collected by police and mortuary staff may be delayed in getting to the entomologist.Live samples may continue developing and alter minimum postmortem interval (PMImin) estimates.This study investigated development of simulated evidential samples of Calliphora vicina Robineau-Desvoidy and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) maggots.Maggots of each species were studied in three developmental classes: "small" (late second/early third instar),"mid" (mid third instar) and "large" (late third instar).Seven replicates of 11 maggots in each class were assigned without food to four treatments: (1) 24 h at 20 ℃;(2) 24 h at 4 ℃;(3) 48 h at 20 ℃ and (4) 48 h at 4 ℃.There was a significant difference in absolute length change across treatments,reflecting size for C vicina,and interaction between size,time and temperature for Ch.rufifacies.Calliphora vicina maggots showed minimal mortality,and most second instar larvae moulted by the experiment's end.Chrysomya rufifacies showed heavy mortality and minimal moulting from the second to third instar.Only "large" Ch.rutifacies maggots kept at room temperature for 48 h pupariated.Since these results confirm that development can continue in live unfed maggot samples after their collection,it is not advisable to delay their preservation.