Age-dependent gene expression of Calliphora vicina pupae (Diptera: Calliphoridae) at constant and fluctuating temperatures.

Estimating the age of the developmental stages of the blow fly Calliphora vicina (Diptera: Calliphoridae) is of forensic relevance for the determination of the minimum post-mortem interval (PMImin). Fly eggs and larvae can be aged using anatomical and morphological characters and their modification during development. However, such methods can only hardly be applied for aging fly pupae. Previous study described age estimation of C. vicina pupae using gene expression, but just when reared at constant temperatures, but fluctuating temperatures represent a more realistic scenario at a crime scene. Therefore, age-dependent gene expression of C. vicina pupae were compared at 3 fluctuating and 3 constant temperatures, the latter representing the mean values of the fluctuating profiles. The chosen marker genes showed uniform expression patterns during metamorphosis of C. vicina pupae bred at different temperature conditions (constant or fluctuating) but the same mean temperature (e.g. constant 10 °C vs. fluctuating 5-15 °C). We present an R-based statistical tool, which enables estimation of the age of the examined pupa based on the analysed gene expression data.

How should living entomological samples be stored?

Sampling and storing insect evidence alive are important tasks in forensic entomology as it can impact survival and growth rates. To investigate the effect of cooling and storing of insect evidence before its arrival in the laboratory, samples of all three larval stages of the blow fly species Lucilia sericata and Calliphora vicina were analyzed. A first group was stored at room temperature and a second one in a refrigerator (~ 5 °C) for 16 h, all without air, supply of food, and sawdust. Afterwards, they were kept at 6-8 °C in a Styrofoam box for 8 h, simulating a transport situation. Mortality rate (MR) was calculated and 25% of the surviving larvae were killed and measured to check for interim growth. The remaining alive specimens were reared at 25 °C until adult's eclosion for estimating a possible storage impact on survival during later development. The results were then compared with a control which was not temporarily stored and chilled but left feeding in boxes with an air-permeable lid on food substrate at 25 °C.A 24-h temporary storage stopped the larval growth in comparison with the control especially in early larval stages in both species. A high MR of up to 100% for third instar (L3) larvae stored both at room temperature and in a cold environment without air supply was found. Oxygen supply can reduce significantly the MR at least for L3 larvae of L. sericata. Findings provide scientific evidence for the recommendation to store larval samples at cold temperatures with both oxygen and food supply. The high MR for samples of the last larval stage clearly shows the need for a fast delivery after sampling and a more sophisticated storage procedure like, e.g., providing air supply. Storing live samples at room temperature without air access should be avoided.

Forensic entomology: applications and limitations.

Forensic entomology is the science of collecting and analysing insect evidence to aid in forensic investigations. Its main application is in the determination of the minimum time since death in cases of suspicious death, either by estimating the age of the oldest necrophagous insects that developed on the corpse, or by analysing the insect species composition on the corpse. In addition, toxicological and molecular examinations of these insects may help reveal the cause of death or even the identity of a victim, by associating a larva with its last meal, for example, in cases where insect evidence is left at a scene after human remains have been deliberately removed. Some fly species can develop not only on corpses but on living bodies too, causing myiasis. Analysis of larvae in such cases can demonstrate the period of neglect of humans or animals. Without the appropriate professional collection of insect evidence, an accurate and convincing presentation of such evidence in court will be hampered or even impossible. The present paper describes the principles and methods of forensic entomology and the optimal techniques for collecting insect evidence.

De novo transcriptome analysis and highly sensitive digital gene expression profiling of Calliphora vicina (Diptera: Calliphoridae) pupae using MACE (Massive Analysis of cDNA Ends).

Determining a post-mortem interval using the weight or length of blow fly larvae to calculate the insect's age is well established. However, to date, there are only a handful studies dealing with age estimation of blow fly pupae, in which weight or length cannot be used as a relevant parameter. The analysis of genetic markers, which indicate a certain developmental stage, can extend the period for a successful post-mortem interval determination. In order to break new ground in the field of age determination of forensic relevant blow flies, we performed a de novo transcriptome analysis of Calliphora vicina pupae at 15 different developmental stages. Obtained data serve as base to establish molecular age determination techniques. We used a new, deeper, and more cost-effective digital gene expression profiling method called MACE (Massive Analysis of cDNA Ends). We generated 15 libraries out of 15 developmental stages, with 3-8 million reads per library. In total, 53,539 distinct transcripts were detected, and 7548 were annotated to known insect genes. The analysis provides high-resolution gene expression profiles of all covered transcripts, which were used to identify differentially expressed genetic markers as candidates for a molecular age estimation of C. vicina pupae. Moreover, the analysis allows insights into gene activity of pupal development and the relationship between different genes interesting for insect development in general.

Forensic entomology in Germany.

Forensic entomology (FE) is increasingly gaining international recognition. In Germany, however, the development of FE has been stagnating, mainly because of the lack of cooperation between police, forensic medicine and entomology. In 1997 a co-operative research project 'Forensic Entomology' was started in Frankfurt/Main at the Center of Legal Medicine and the Research Institute Senckenberg. The aim of this project is to establish FE in Germany as a firmly integrated component of the securing of evidence from human cadavers in cases of suspected homicide. For this purpose we developed a forensic insect collecting kit, and policemen are educated for greater acceptance and better application of FE. The scientific programme focuses on the investigation of the insect succession on cadavers in urban and rural habitats. This also includes new indicator groups (e.g. parasitic wasps) for a more precise calculation of the late post mortem interval. Recently a DNA-based reliable and fast identification method especially for the immature stages of necrophagous insects became part of the project. Preliminary results are reported and two case studies presented.

The history of forensic entomology in German-speaking countries.

Megnin's book "La fauna des cadaveres" published in 1894 in France is generally accepted as a mile-stone in forensic entomology. It is hardly known that at the same time this topic was likewise explored in the German-speaking countries. Even PMI estimation based on developmental data of blowflies was performed. After a more descriptive period in the first half of the 20th century the complexity and variability of insects' biological behavior were detected and formally investigated. Improved technical facilities, enhanced comprehension of scientific studies and multidisciplinary cooperation, enabled rapid progress in forensic entomology during the last decades. With the European Association for Forensic Entomology founded in 2002 the frame work for a high standard of competency at an international level was constituted.

Paternity-testing on paraffin-embedded abortion tissue: preparation of fetal cells may be indispensable.

In a case of sexual abuse, a paternity test was performed on paraffin embedded abortion material. STR typing was successful only after isolating fetal tissue from the abortion-material and separately extracting DNA from the excised fetal cells. Examination with five STRs led to a paternity index of 332, confirming the abuse that had resulted in pregnancy.

Population genetic study of the simultaneously amplified loci HLA DQA1, LDLR, GYPA, HBGG, D7S8, and GC in a German population sample.

A population genetic study of the HLA DQA1 and the "Polymarker" loci LDLR, GYPA, HBG, D7S8, and GC has been performed in a German Caucasian population (Frankfurt am Main area). All loci are in Hardy-Weinberg equilibrium and no unexpected association of loci has been observed. The data of the allele distributions are similar to those of other Caucasian populations. All six loci together have a power of discrimination (PD) of 0.9996 and an exclusion chance in paternity testing of 0.81.

Evaluation of a rapid assay system, HIV 1/HIV 2 Testpack, Abbott, to detect human immunodeficiency virus antibodies in postmortem blood.

For evaluating the HIV 1/HIV 2 Testpack (Abbott, Chicago, IL) to detect antibodies to human immunodeficiency virus (HIV) in whole postmortem blood 456 samples were collected prior forensic autopsies. All samples were tested using the enzyme-linked immunoassay (ELISA) and the Testpack; positively reactive samples and samples with equivocal results were confirmed by Western blot. Of the 456 samples 21 (4.6 per cent) proved to be reactive in both systems (confirmed by Western blot). In 17 cases (3.7 percent) interpretation of the result was difficult, but no serious misinterpretations occurred. It is concluded that the HIV-Testpack provides accurate results in testing whole postmortem blood for HIV antibodies.

[Attempted fraud in legal identity determination--detection by DNA single locus analysis]. / Betrugsversuch bei Identitätsgutachten--Aufdeckung durch DNA-Single-Locus-Analyse.

In a case where a mix up of blood samples was claimed, an identity check was carried out by means of four single-locus-probes. DNA analysis produced identical profiles with two of the probes (MS 31 and MS 43). With the two other probes one matching and one differing fragment could be detected respectively (MS 1 and g3) In addition to the unambiguous statement "no identy", the DNA analysis proved that the tested blood samples must have been those of two closely related people, most probably of brothers and sisters. Attempted fraud can be assumed from these findings. Further investigations and additional DNA analysis which included a blood sample of the brother of the accused, revealed that the sample for the identity check had been taken from the brother and not from the accused himself.

The nocturnal oviposition behaviour of blowflies (Diptera: Calliphoridae) in Central Europe and its forensic implications.

Numerous factors may cause delayed colonisation of a corpse by blowflies, leading to a discrepancy between the entomologically determined post-mortem interval (PMI) and the time of death. Blowflies, for example, are considered to be inactive at night, however, published observations are contradictory. In the present study, several field experiments and one type of indoor experiment were conducted in summer of 2004 and 2005 in order to investigate the nocturnal ovipositional behaviour of blowflies. In the field, two types of bait, dead hedgehogs and fresh beef liver, were placed at night in different urban and rural locations in Frankfurt and in Munich, Germany. For the indoor-experiments beef liver was placed in small plastic boxes containing caged Lucilia sericata females in the evening and left overnight. At night, no ovipositon was observed in the field (n=51, T=10-24 degrees C). Nocturnal oviposition in complete darkness occurred in the plastic boxes in two of six cases (T=25 degrees C). Considering the behavioural and physiological characteristics of flies we suggest that nocturnal oviposition of blowflies appears to be unlikely under natural conditions in Central Europe but may occur under certain circumstances, such as unusual high nightly temperatures and the presence of gravid flies with an appropriate arousal threshold.

DYS19 and amelogenin in artificial blood stains with defined amounts of male and female cells.

The sensitivity of the DYS19 and the amelogenin STR systems for amplifying Y-specific fragments was assayed using artificial bloodstains with varying amounts of male and female (non-template) DNA in different ratios. The study confirmed the high sensitivity of both systems in detecting male-specific PCR fragments in stains containing 10-25 template molecules even in the presence of large amounts of female DNA in the mixture by silver-stain detection. However, blood mixtures which contain less than 10% male cells could be reliably typed only when at least 100 template molecules were present in the artificial bloodstain, due to increasing amounts of hemoglobin from the female blood which is a PCR inhibitor.

RFLP and sequence analysis of the cytochrome b gene of selected animals and man: methodology and forensic application.

To identify common animal species by analysis of the cytochrome b gene a method has been developed to obtain PCR products of a large domain of the cytochrome b gene (981 bp out of 1140 bp) in humans, selected mammals and birds using the same specifically designed primers. Species-specific RFLP patterns are generated by co-restriction with the restriction endonucleases ALU I and NCO I. The RFLP patterns obtained are conclusive even in mixtures of two or more species. The results were confirmed by sequence analysis which in addition explained intraspecies variations in the RFLP patterns. The method has been applied to forensic casework studies where the origin of roasted meat, stomach contents and a bone sample has been successfully identified.

Characterization of a highly variable short tandem repeat polymorphism at the D2S1242 locus.

We report the evaluation of short tandem repeat (STR) locus D2S1242 (GDB ID G00-309-429) for forensic purposes, investigated by polymerase chain reaction (PCR) amplification and both native and denaturating polyacrylamide gel electrophoresis in 147 unrelated Austrians. No deviations from Hardy-Weinberg expectations were observed. The mean exclusion chance (MEC) was 0.669, the discriminating power (DP) was 0.947, and the observed heterozygosity rate was 0.856. An allelic ladder consisting of eight sequenced alleles (141-167 and 175 bp) was constructed. Sequence analysis revealed that the locus comprised two repeat motifs varying in number between alleles GAAA and GAAG. According to the number of tetranucleotide repeats the smallest allele was designated as 10 and the largest allele as 18.

[Piperacillin: pharmacokinetic studies on the biliary and renal excretion (author's transl)]. / Piperacillin: Kinetische Studien über biliäre und renale Ausscheidung.

9 patients (6 female, 3 male) with either cholelithiasis or choledocholithiasis submitted to cholecystectomy were studied during the postoperative period. Sodium 6-[D-(--)-alpha-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-alpha-phenyl-ace tamido]penicillinate (piperacillin) was administered in single i.v. doses of 2 and 4 g on the 4th and 6th postoperative days. Serum samples were taken at intervals of 20 min over a period of 3 h post injection and bile was collected via T-tube drainage in 20-min periods also for 180 min. The total urine outputs were collected for the periods 0--6 h, 6--12 h and 12--24 h after administration of the drug. The maximum concentration of piperacillin in the bile was observed 60--120 min post administration. The values in the bile were found to exceed the corresponding serum concentrations 30--40fold. Within 3 h 9.6 (+/- 6.5)% and 13.4 (+/- 5.0)% were eliminated in the bile after 2 and 4 g injections, respectively. The percentage of the administered drug excreted in the urine within 24 h was 56.3% (2 g i.v.) and 66.0% (4 g i.v.).

Effects of cycloSal-d4TMP derivatives in H9 cells with induced AZT resistance phenotype.

Cytotoxic and antiretroviral activity of cycloSal-d4TMP derivatives were tested in a new AZT-resistant H9 cell subline (H9rAZT250). The results showed, that cycloSal-d4TMP derivatives overcame resistance of HIV-1 to d4T in H9rAZT250 cells, which exert decreased thymidine kinase (TK) gene expression.