Effect of temperature on development of Liopygia (= Sarcophaga) argyrostoma (Robineau-Desvoidy) (Diptera: Sarcophagidae) and its forensic implications

The temperature-dependent development of the forensically important flesh fly Liopygia (Thomsonea) argyrostoma (Robineau-Desvoidy) (=Sarcophaga argyrostoma) was studied at six constant temperature regimes in the laboratory. Total developmental time of L. argyrostoma from larviposition to adult emergence was 54.9+/-1.45, 31.3+/-1.1, 22.2+/-0.67, 16.3+/-0.54, and 14.9+/-0.4 days (+/-S.D.) when reared at 15, 20, 25, 30, and 35 degrees C, respectively. At 8 degrees C, larval development was not completed. From linear regression of development rates of five of the six studied constant temperature regimes, it was determined that the minimum development threshold (tL) for total immature development is 7.4 degrees C, and the overall thermal constant (K) for L. argyrostoma is 396.4+/-19.18 (mean +/- S.D.) day-degrees (DD) above the threshold.     

The development of the black blow fly, Phormia regina (Meigen).

The black blow fly, Phormia regina (Meigen) is a primary species commonly utilized to indicate a postmortem interval, or more appropriately a "time since colonization". Due to the importance of this species as a secondary myiasis producer in livestock operations, and more recently as a time since death indicator in the field of forensic entomology, a considerable amount of data on its growth and development has been generated. However, the developmental time as reported by these studies varies greatly, and current more detailed data is needed for use in medicocriminal entomology. Hourly developmental data is presented under constant temperatures of 10, 15, 20, 25, 30, 35 and 40 degrees C, and cyclic temperatures of 10-15, 15-25, 25-35 and 35-45 degrees C. This study is in agreement with the results reported by Kamal [Comparative study of thirteen species of sarcosaprophagous Calliphoridae and Sarcophagidae (Diptera). I. Bionomics, Ann. Entomol. Soc. Am. 51 (1958) 261] and Melvin [Incubation period of eggs of certain musciod flies at different constant temperatures, Ann. Entomol. Soc. Am. 27 (1934) 406] only at temperatures of 25 degrees C and below. Bishopp [Flies which cause myiasis in man and animals: some aspects of the problem, J. Econ. Entomol. 8 (1915) 317] reported a shorter developmental duration for larval stages than what was produced with our laboratory rearings.     

Effect of temperature on development of the forensically important holarctic blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae)

Immature development times of the blow fly Protophormia terraenovae (Robineau-Desvoidy, 1830) were studied in the laboratory at five different constant temperatures (15, 20, 25, 30, 35 degrees C). The minimal duration of development from oviposition to adult emergence was inversely related to temperature, ranging from 9.19+/-0.3 days at 35 degrees C to 37.78+/-2.96 days at 15 degrees C. From linear regression of development rates at the five studied constant temperature regimes, it followed that the minimum development threshold (t(L)) for total immature development is 8.95 degrees C ( approximately 9 degrees C) and the overall thermal constant (K) for P. terraenovae is 240.2+/-9.3 day-degrees (DD) above the threshold. Linear regression of developmental rates from oviposition to pupariation resulted in a minimum development threshold of 9.8 degrees C. However, it is possible that developmental time from oviposition to adult eclosion might be different in various regions of the world, and that the thermal constant of a holarctic species like P. terraenovae is not same everywhere. Additionally, as the present paper shows, studies characterizing variation in these parameters between geographically distinct populations of the same species would be of great value for future forensic entomological casework.     

Effects of refrigeration on the biometry and development of Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae) and its consequences in estimating post-mortem interval in forensic investigations

The aim of this study was to simulate the low temperatures that insects could experience between the time being sampled from cadavers and their arrival in the laboratory. This was in order to investigate the effect of low temperature on development of maggots. At different stages of development, individuals of Protophormia terraenovae (Robineau-Desvoidy) reared at 24 degrees C were submitted to a temperature of 4.0+/-0.5 degrees C for a period varying from 1 to 10 days. Independent of the stage of development at which the insects were refrigerated, the treatment induced significant changes on the duration of development. The effect of low temperature on the developmental time between the return to 24 degrees C to adult emergence depended on the larval stage that was refrigerated. When first instar larvae and prepupae were refrigerated, the time to emergence at 24 degrees C decreased with an increase of duration of the refrigeration period. Time to emergence increased under the same conditions when second instar larvae and pupae were refrigerated. These results indicate that keeping larvae of P. terraenovae at 4 degrees C does not just simply lead to a cessation of metabolism but disturbs the regular development. Ten days of cooling induced an error in estimating post-mortem interval (PMI) of more than 6h.     

Bionomics of two forensically important blowfly species Chrysomya megacephala and Chrysomya putoria (Diptera: Calliphoridae) reared on four types of diet

The use of heterogeneous animal tissues for the rearing of necrophagous insect species can produce uneven biological data, which can compromise the determination of larval age and, consequently, estimates for post-mortem intervals. We investigated the development of two species, Chrysomya megacephala and Chrysomya putoria (Diptera: Calliphoridae), reared on four substrates: minced beef (control) and semi-synthetic diets with the addition of sardine, rumen or chicken eggs. No differences in total developmental times were detected among larvae reared on different diets. Length and width of larvae were partially affected by the type of food. Third instar larvae and pupae of both species were heavier on beef treatment when compared with other substrates. Overall mortality was lower when beef was used as food. Longevity of adults and sex ratio were not negatively affected by the use of diets. Egg-based diet was the least effective for both species. Given the fact that several bionomical parameters of larvae reared on diets were close to those obtained when minced beef was offered, and considering the putrid odour, frequency of contamination and lack of homogeneity of animal tissue, semi-synthetic diets can be used for rearing C. megacephala and C. putoria.     

Growth behavior of the blue blowfly Calliphora vicina maggots

Fifty-three clusters of blowfly eggs of the genus Calliphora vicina were observed in the laboratory up to the hatching stage under reproducible and virtually field-like conditions. Rearing the larvae was then continued up to pupation, the larval growth in length being recorded several times a day. As the object was to study the dependence of the larvae increase in length on the temperature conditions in vitro, the substratal humidity and food supply were kept unchanged during the entire study. The temperature ranged from 6.5 degrees C to 35 degrees C, with the temperature for the individual cluster kept constant during the entire developmental process. Data on about 5500 measured larvae were statistically evaluated. The basic result established was that in the case of the blowfly of the genus Calliphora vicina in vivo, all developmental stages relevant to the entomologic determination of the time of death depend on the temperature conditions: (1) the duration of the egg stage increases with decreasing temperature; (2) the speed of larval growth is slower at lower temperatures; (3) the maximal larval length is reached earlier at higher temperatures; (4) the mean value of maximal length decreases with increasing temperature; (5) larvae under all temperature conditions decrease in size after having reached their maximal length, the decrease in length being more rapid at higher temperatures; (6) constant temperatures over 30 degrees C lead to "stunted forms" which do not pupate and die; (7) constant temperatures under approximately 16 degrees C after the peak of growth has been reached inhibit the readiness to pupate, which causes the larvae to fall into a stationary state of rest, which will be interrupted only when the temperature is raised and resumption of the metamorphosis is thus induced. To allow rapid reconstruction of the larval age in general practice, the established growth data were set out in the form of a diagram designated isomegalendiagram, which permits temperature-fluctuation-related entomologic determination of the time of death with a maximum degree of accuracy.     

Effect of Temperature on Six Different Developmental Landmarks within the Pupal Stage of the Forensically Important Blowfly Calliphora vicina (Robineau‐Desvoidy) (Diptera: Calliphoridae)

This study investigates the pupal development times of the blow fly Calliphora vicina, which were studied in the laboratory at six different constant temperatures (15, 20, 23, 25, 28, and 30°C each ± 1°C). Lower thresholds (t_L) for development were estimated from the linear regression of the developmental rates on each temperature. These data have made it possible to calculate the accumulated degree days (ADD) necessary for C. vicina to complete the larval stage and to achieve adult emergence. The minimal duration of development from oviposition to adult emergence was found to be inversely related to temperature. Additionally, six landmarks in pupal development are showed, and for each of the landmarks, the ADD value was calculated for every rearing temperature involved. These data assist in calculating the duration of the pupal stage based on morphological characteristics and would be of great value for future forensic entomological casework.     

Thermal requirements of immature stages of Chrysomya albiceps (Diptera: Calliphoridae) as a common forensically important fly

Entomological material may be used to estimate the time since death occurred (postmortem interval, PMI) in forensically obscure cases. The method that is commonly used to calculate minimum post-mortem interval (mPMI, i.e., the least amount of time since one can be confident death occurred) is based on the relationship between insect development and ambient temerature. Isomegalen and isomorphen diagrams are among methods allowing to calculate the age of necorphagous insects, yet thermal summation models provide the most precise and acurate estimations. The digrams are prepared based on the length or the developmental stages of the larvae as a function of time and mean ambient temperature. A knowledge of thermal requirements, in particular lower temperature threshold (D_z) at which development of a species terminates, is of essential importance to calculate ADD (Accumulated Degree Days). In this study different temperature regimes were used to construct the isomorphen diagram, examinate changes in larval body length at different ambient temperatures and to estimate the thermal requirements for developemnt of Chrysomya albiceps, the most common dipteran species reported on human and animal cadavers in Iran. Six development events including hatching, 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion were studied under eleven constant temperature regims (17–37 ⁰C). The development rate of Ch. albiceps increased as temperature increased. The larval length peaked at the end of third stage and then decreased at wandering stage. The maximum larval length occurred at 72 h post oviposition at either 31, 33, or 35 °C. At 17 °C, larvae did not hatch from eggs and at 37 °C wandering larvae did not proceed to pupariation, and thus larval development were analysed at the nine left over temperatures. The development stages required at least (D_z ± SE) 13.04 ± 0.37, 14.29 ± 0.45, 15.69 ± 0.56, 15.18 ± 0.56, 14.94 ± 0.48, and 11.23 ± 0.41 °C to reach one of the successive developmentl events, respectively. The estimated thermal summation constant (k) for those the six events were 10.43 ± 0.27, 19.31 ± 0.32, 27.87 ± 1.3, 55.94 ± 1.82, 66.69 ± 3.5, and 143.52 ± 5.61 ADD accordingly.     

Temperature-correction of abdominal impedance: improved relationship between impedance and postmortem interval

The relationship between extracellular abdominal impedance and postmortem interval (PMI) reflects the combined effects, on impedance, of postmortem cooling of the tissues and of autolysis per se. This study was performed in order to eliminate temperature change as a major factor contributing to the time course of postmortem change in abdominal impedance. Dissociation of thermal and autolytic influences was achieved by recording deep abdominal temperature at the time of impedance measurement, followed by correction of all measured impedances to their theoretically predicted values at an arbitrarily chosen temperature of 40 degrees C. Uncorrected abdominal impedance increased from 82+/-12 Ohmz, 1 h after death, to 108+/-21 Ohmz after 12 h. Impedance then decreased to 96+/-23, 89+/-22, 75+/-19, 66+/-21 and 59+/-19 Ohmz at postmortem intervals of 24, 36, 48, 60 and 72 h, respectively. In contrast, corrected abdominal impedance decreased progressively from 63+/-7 Ohmz, 1 h after death, to 61+/-9, 56+/-11, 51+/-10, 46+/-10, 39+/-11 and 35+/-10 Ohmz at postmortem intervals of 12, 24, 36, 48, 60 and 72 h, respectively. The improved relationship between (corrected) abdominal impedance and PMI is of potential value in estimating time since death.     

Influence of substrate tissue type on larval growth in Calliphora augur and Lucilia cuprina (Diptera: Calliphoridae)

The size of fly larvae is an important variable in the use of these insects to estimate postmortem interval. Furthermore, the nutritional intake of larvae is likely to vary subject to the part of a corpse on which they are feeding. A study was therefore conducted to investigate the effect of type of food substrate on larval growth in two species of forensically important Australian blowflies. After collection on sheep's liver in the laboratory, different groups of larvae of Lucilia cuprina (Wiedemann) and Calliphora augur (Fabricius) were grown on sheep's liver, meat, and brains, and their body lengths compared. Results indicated that the development of larvae fed sheep's liver was adversely affected compared with larvae fed meat and brain; they moulted later, reached maximum length more slowly and sometimes produced significantly smaller pupae. These findings, similar to those of another recent study, have obvious implications for postmortem interval determinations. Estimates may be considerably skewed if the site of collection of larvae at a death scene contains tissue types different to those used in reference experiments. We therefore recommend caution in forensic analyses that interpret crime scene data using developmental studies performed with a single type of larval food substrate.     

Insects of forensic significance in Argentina

Records from forensic expertises and trappings with beef baits conducted in Buenos Aires, Argentina (34 degrees 36'S), show that the dominating species are widespread ones (Calliphora vicina and Phaenicia sericata), with different behaviour in each large latitudinal zone. It is suggested that the range of the yearly photoperiod variation has an influence in the behaviour of the blowflies, making up for differences in the succession patterns. The Calliphorid blowflies Cochliomyia macellaria and Chysomya albiceps were found on indoors corpses; the latter also on outdoors corpses when blood was shed, and in that case as primary. Three species of beetles of the genus Dermestes, which had been associated with mummified remains, appeared 10-30 days after death. The Silphid beetle Hyponecrodes sp. cf. erythrura was found on outdoor copses in rural environments. The Nitidulid beetle Carpophilus hemipterus was found in association with the cheese skipper Piophila sp. (Diptera: Piophilidae) in medullar cavities of bones after ca. 30 days; to this association is often added the Clerid Necrobia rufipes. Lepidoptera Tineidae appear on the head of mummified indoors corpses. North of parallel 32 degrees S, the Muscid grave-fly Ophyra sp. was found breeding on a corpse outdoors in summer. A division by latitude and climate is proposed for Argentina, and an extended system is proposed for the world.     

Peroxidase activity in traumatic skin lesions

Peroxidase activity was determined in experimental compression-excoriation lesions and incision wounds of rat skin after different periods of vital time. The peroxidase enzyme was extracted from the tissues by homogenization in 0.5% cetyltrimethylammoniumbromide, and the enzyme activity was measured from the supernatant by o-dianisidine-H2O2 assay. In the blood of the rats a mean activity of approx. 5.26 +/- 1.11 U/g dry weight was observed. In the control specimens of the skin the activity was very low and generally below the detection limit of the methods used. In 30-min-old compression-excoriation lesions the mean peroxidase activity was 0.38 +/- 0.21 U/g dry weight. In lesions older than 30 min the activity started to increase rapidly. In 4-h-old compression-excoriation lesions it was 10 times higher than the 30-min level and was 40 times higher in 12-h-old lesions and 70-100 times higher in 1-3-day-old compression-excoriation lesions, respectively. In 30-min-old incision wounds the mean peroxidase activity was 0.65 +/- 0.37 U/g dry weight. The increase of the activity compared with the 30-min level was even faster in the incision wounds: in 4-h-old wounds the mean activity was 50 times higher, in 12-h-old wounds 200 times higher and in those of 1-5 days it was several hundreds of times higher. Compression-excoriation lesions made after death showed activity similar to the control specimens. Postmortem autolysis at +22 degrees C resulted in a loss of the enzyme activity in 1-day-old compression-excoriation lesions so that after 3 days approx. 80% remained, and after 5 and 7 days approx. 40% was present. After 3 days of autolysis at +4 degrees C, nearly 100% of the activity remained and approx. 90% was present after 5 and 7 days of autolysis. Increased peroxidase activity was also detectable in human vital excoriations in the specimens which were taken in autopsies several days postmortem.     

Larval-mass effect: Characterisation of heat emission by necrophageous blowflies (Diptera: Calliphoridae) larval aggregates

Numerous Calliphoridae species larvae are necrophageous and develop on animal cadavers. During the feeding stages, a strong gregarious behaviour leads to the formation of large larval masses, allowing larvae to share digestive fluids. Furthermore, a mass of larvae emits heat, resulting in a local increase of temperature. Differences greater than 20°C between ambient and larval mass temperatures have already been observed, and the temperature of the mass can reach 50°C. Thus, larvae could benefit from this increased local temperature to speed up their development. This study focuses on the dynamic and characterisation of heat emission by Lucilia sericata (Diptera: Calliphoridae) (Meigen, 1826) larval masses. Experiments were performed under controlled conditions using several ambient temperatures and different numbers of larvae. Results indicate that heat emission depends on the instar and is strongly affected by available amount of food. Furthermore, according to the experimental data, the heat emission is also relative to the weight of the larval mass, the larvae number and the local temperature. These results also demonstrate that optimum ambient-temperature values ranging between 22°C and 25°C produce maximal heat-emission per larva. Furthermore, a feedback loop, involving heat exchanges and physiological and behavioural thermoregulation processes, appears inside aggregates. In the context of forensic cases and post-mortem interval (PMI) estimation, these results indicate that heat emission can occur even with "small" masses if they are composed of high number of second stage larvae. Furthermore, particular attention should be paid on cases involving L. sericata larvae at ambient temperature ranging from 22°C to 25°C, which appears to maximise the heat-emission process.     

Validation of male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.     

Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram.

Developmental behavior of eggs, larva and pupa of the blowfly species Lucilia sericata (Meigen) were studied under 10 different temperature regimes. Data from these studies were used to construct the isomegalen-diagram. In this diagram, time from hatching to peakfeeding is plotted against temperature, each line representing identical larval length at various temperatures. If the temperature is roughly constant, as is the case with corpses found indoors, the age of the maggot can be read off instantly from its length, provided that the maggot has not entered the migratory phase. Where temperature is variable, an age range can be estimated between the points where the measured larval length cuts the graph at the maximum and minimum temperatures recorded. Equally, the isomorphen-diagram representing all morphological stages from oviposition to eclosion should be used, if maggots in the migratory phase or pupae or puparia are recovered from the scene. The isomegalen- and the isomorphen-diagrams could facilitate a quick and more precise estimate of the postmortem interval even for the inexperienced investigator. In addition, our results vary from those of other investigators, suggesting a different thermal behavior of the holarctic blowfly L. sericata in various zoogeographic regions.     

Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.     

Methods used for the killing and preservation of blowfly larvae, and their effect on post-mortem larval length

A record of the length of the largest larvae collected from a corpse can be used to estimate the age of the oldest larvae present and, therefore, give an estimate of minimum time since death. Consequently, factors that affect post-mortem larval length will impact on any estimate of PMI based on it. Methods used to kill and preserve larvae are known to affect post-mortem length. This study looks at the effects of different preservatives, and variations in the protocol used for killing larvae by immersion in a hot water bath = [hot water killed; HWK], on the length of dead larvae of two common blowfly species. Post-feeding third instar Calliphora vomitoria and Lucilia sericata larvae were either HWK in boiling water and then placed in 80% ethanol or 10% formaldehyde solution, or placed live into the preservatives. For both species, choice of preservative and method of killing significantly affected post-mortem length. There were significant interspecific differences in their response to identical methods of killing and preservation. Additional experiments were carried out where C. vomitoria larvae were HWK in water at 80 and 100 degrees C for 1, 30, 60 and 90 s duration. Both temperature and duration significantly affected post-mortem length. Maximum length was attained after at least 60 s immersion. The amount of post-mortem decomposition that occurred after the larvae were placed in preservative could be greatly reduced by increasing the duration of immersion and/or increasing the water temperature. For the HWK larvae, it was possible to record their length immediately after death and before they had been placed in preservative. This data revealed that where 80% ethanol was used as a preservative the larvae expanded in the preservative. The timing of this expansion was investigated with a sample of C. vomitoria, HWK at 100 degrees C for 30 s and recording post-mortem length immediately after death and again after 3, 6, 9, 12, 24, 27, 30 and 33 h storage in 80% ethanol. Maximum length was recorded after 12 h storage and the rate of expansion was highest during the first 3 h in this preservative. After long-term storage (290 days), larvae killed and preserved in the same way were on average 0.7% longer than immediately after death and 0.6% (0.11 mm) smaller than when last measured (after 28 days storage).     

Diurnal and Nocturnal Flight Activity of Blow Flies (Diptera: Calliphoridae) in a Rainforest Fragment in Brazil: Implications for the Colonization of Homicide Victims

Nocturnal flight of blow flies (Diptera: Calliphoridae) is a controversial issue in forensic entomology. We performed two field experiments to investigate the diurnal and nocturnal activity of six blow fly species in a rainforest fragment in Brazil. Initially, nocturnal (17:30–05:30) versus diurnal (05:30–17:30) flight activity was investigated. Only 3.9% of adults were collected at night, mostly the native species Mesembrinella bicolor, and nocturnal oviposition did not occur. In the second experiment, collection of adults took place at the following intervals: 05:30–08:30, 08:30–11:30, 11:30–14:30, and 14:30–17:30. The proportions of adults did not differ significantly among the four diurnal intervals, except for Hemilucilia segmentaria, which was captured more frequently in the early morning. Calliphoridae has predominantly diurnal behavior, not laying eggs in darkness. The association of the native species M. bicolor, Hemilucilia semidiaphana, and H. segmentaria to forested areas reinforces the forensic relevance of data on their flight pattern.     

The succession and development of insects on pig carcasses and their significances in estimating PMI in south China

The succession of insect communities on carrion varies at local and global spatial scales. As such, ecological succession data obtained from corpses at one geographic location cannot necessarily be applied to other locations. Our study describes this succession in the far southern part of China to provide such data for forensic cases in this region. A total of 18 pig carcasses were placed in the field in four seasons, and the timing of the following events were recorded: appearance of larvae, onset of larval wandering, when most larvae had wandered, onset of pupariation, when most larvae had pupariated, onset of eclosion and end of eclosion. Our results indicated that all of the evaluated events could be used as accurate indicators of postmortem interval (PMI). The carcasses decayed fairly quickly in spring, summer and autumn, taking 225+/-75 h, 183+/-44 h, and 247+/-70 h, respectively, to decay from the fresh stage to skeletonisation. In winter, carcasses needed longer (1180+/-291) to decay as much. Carcasses attracted 47 species of insect, with flies predominating. The larvae were mainly Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart) and Hydrotaea (Ophyra) spinigera (Stein). Most necrophagous insects were found all year around, and there were no marked differences in species among the four seasons, except that Dermestes maculatus (De Geer) was absent in winter. Blowflies produced only one generation on a carcass before it became skeletonised, which simplified the estimation of PMIs.     

Stability study of the designer drugs "MDA, MDMA and MDEA" in water, serum, whole blood, and urine under various storage temperatures.

A controlled study was undertaken to determine the stability of the designer drugs MDA, MDMA and MDEA in pooled serum, whole blood, water and urine samples over a period of 21 weeks. The concentrations of the individual designer drugs in the various matrices were monitored over time, in the dark at various temperatures (-20, 4 or 20 degrees C), for a low (+/- 6 ng/ml for water, serum and whole blood and +/- 150 ng/ml for urine) and a high concentration level (+/- 550 ng/ml for water, serum and whole blood and +/- 2500 ng/ml for urine). Compound concentrations were measured using a validated HPLC assay with fluorescence detection. Our study demonstrated no significant loss of the designer drugs in water and urine at any of the investigated temperatures for 21 weeks. The same results were observed in serum for up to 17 weeks, and up to 5 weeks in whole blood. After that time, the compounds could no longer be analyzed due to matrix degradation, especially in the low concentration samples that were stored at room temperature. This study demonstrates that the designer drugs, MDA, MDMA and MDEA are stable when stored at -20 degrees C for 21 weeks, even in haemolysed whole blood.