Discrimination of Diptera order insects based on their saturated cuticular hydrocarbon content using a new microextraction procedure and chromatographic analysis.

The nature and proportions of hydrocarbons in the cuticle of insects are characteristic of the species and age. Chemical analysis of cuticular hydrocarbons allows species discrimination, which is of great interest in the forensic field, where insects play a crucial role in estimating the minimum post-mortem interval. The objective of this work was the differentiation of Diptera order insects through their saturated cuticular hydrocarbon compositions (SCHCs). For this, specimens fixed in 70 : 30 ethanol : water, as recommended by the European Association for Forensic Entomology, were submitted to solid-liquid extraction followed by dispersive liquid-liquid microextraction, providing preconcentration factors up to 76 for the SCHCs. The final organic extract was analysed by gas chromatography coupled with flame ionization detection (GC-FID), and GC coupled with mass spectrometry was applied to confirm the identity of the SCHCs. The analysed samples contained linear alkanes with the number of carbon atoms in the C9-C15 and C18-C36 ranges with concentrations between 0.1 and 125 ng g-1. Chrysomya albiceps (in its larval stage) showed the highest number of analytes detected, with 21 compounds, while Lucilia sericata and Calliphora vicina the lowest, with only 3 alkanes. Non-supervised principal component analysis and supervised orthogonal partial least squares discriminant analysis were performed and an optimal model to differentiate specimens according to their species was obtained. In addition, statistically significant differences were observed in the concentrations of certain SCHCs within the same species depending on the stage of development or the growth pattern of the insect.

In vitro comparison of two food choice methodologies for necrophagous species of the genus Thanatophilus (Coleoptera: Staphylinidae: Silphinae).

To accurately model the food webs, we need to acquire precise data on food ecology of the interacting species. This allows better understanding of the trophic interactions and for the necrophagous species this information could be used in medico-legal investigations. For this reason, we recently proposed standardized laboratory methodology to assess the foraging strategies based on parallel testing of 2 food items (meat, dead larvae) (Jakubec et al. 2021). The original methodology had 2 shortcomings. It was not suited for testing living larvae, which could prove predatory behavior of the species. The methodology was also based on parallel experimental design, where the food items are tested together, which could underestimate the maximum consumption of the tested subject for some items. To test if these concerns are valid, we improved original methodology allowing testing living larvae as well as a new sequential experimental setup, where consumption of each item is tested individually in a random order, thus theoretically giving an unbiased maximum consumption estimate. These methodologies were tested head-to-head on 3 forensically relevant species from the genus Thanatophilus (Thanatophilus micans (Fabricius 1794)(Fabricius 1794), Thanatophilus rugosus (Linnaeus, 1758), and Thanatophilus sinuatus(Fabricius, 1775)). The experiments have confirmed that all 3 species are almost strictly necrophagous, although they were capable of predation, despite the presence of preferred food (meat). The comparison also showed that the sequential design has indeed improved capability to quantify the maximal consumption of the given food item. Thus, we suggest following this methodology in future studies.

Initial Work on Sex-Specific Growth Curve Guidelines for Forensically Important Blow Flies Using Chrysomya megacephala (Diptera: Calliphoridae) Entire Age Cohorts.

Sex is a factor influencing development in many insect species, but is not widely studied in forensically important blow flies. If sex influences blow fly development, knowing the sex of a larva from a corpse can increase precision in estimates of that larva's age. The improved prediction of larval age will make estimates of time since death using entomological evidence better. Larvae lack sexually dimorphic morphological characteristics, so sex is not immediately known visually. To generate sexually dimorphic reference growth curves, a subsample must be large enough to ensure enough males and females are present for comparison. Using two entire age Chrysomya megacephala (Fabricius) cohorts, we evaluated the minimum sample number needed to have enough individuals of both sexes for comparison using 95% prediction intervals. Through a simulation of three trials of 1000 random replicates, we determined that a sample size of 19 would prevent any instance of a comparison not occurring because of insufficient sampling from one sex. As the current method for molecular sex determination can be expensive, we also compared how the results of various subsampling percentages compare those of the entire age cohorts. We found that subsampling at least 50% of an entire cohort leads to almost identical results in comparison to the entire age cohort. Together, these findings will help establish guidelines for generating sex-specific reference growth curves. A uniform approach to generating these sex-specific growth curves will lead to more consistency in age estimates made from them.

Developmental models of the carrion beetle Thanatophilus rugosus (Linnaeus, 1758) (Coleoptera: Silphidae).

Coleoptera are currently considered a fundamental tool to help solve criminal investigations, allowing forensic entomologists to estimate post-mortem intervals and obtain other ecology-related information. Thanatophilus rugosus (Linnaeus, 1758) is an important necrophagous beetle distributed through most of the Palaearctic region, where it is readily found on human bodies and animal carcasses. In this study, the new thermal summation models for all the developmental stages of Thanatophilus rugosus are provided. Beetles were reared at six different constant and ecologically relevant temperatures (12, 14, 16, 18, 20, and 22 °C), and their developmental times were measured. Thermal summation constants were calculated for each developmental stage (egg, three larval instars, post-feeding stage, and pupa).

Effect of constant and fluctuating temperature on the intrapuparial development of Sarcophaga argyrostoma (Robineau-Desvoidy, 1830; Diptera: Sarcophagidae).

This study aimes to investigate the effect of constant and fluctuating temperatures on starting times of developmental periods of insect species. For this purpose, Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830), a forensically important species has been chosen. Whether the constant (25°C) and fluctuating (25.2°C averaged over natural conditions) temperatures would make a difference on the timing of intrapuparial development of this species was investigated. First larval instars were placed in rearing cages until they reached post-feeding third larval instar, which is considered the beginning of intrapuparial development. Relevant morphological changes were observed by hourly dissections. At fluctuating temperature treatment, the post-feeding third instar larvae were transferred to containers under natural, open-air conditions. Minimum and maximum starting times of 23 intrapuparial development periods were determined for both treatments. These times, medians, and standard deviations were compared by Mann-Whitney U tests. Accumulated degree hour values were also calculated for these periods. Regardless of temperature treatment, intrapuparial development periods were similar and adult emergence was observed at 289.5 ( ± 1.52) h under 25°C constant temperature and 319 ( ± 1.41) h under fluctuating temperature conditions. No statistical difference was found between the intrapuparial development of S. argyrostoma reared under constant and fluctuating temperatures, except "pupal-adult apolysis" and "checkered pattern on the abdomen becoming distinct" periods. The use of the results obtained when determining the effect of constant and fluctuating temperatures on the starting times of developmental periods of this species in forensic entomology, is also important in transferring developmental biology data into the field of applied sciences.

The paradigm of interdisciplinarity in forensic investigation. A case in Southeastern Spain.

The paper presents the procedure followed to fully solve a forensic case which occurred in Southeastern Spain through the collaboration of specialists from different scientific areas belonging to two different government institutions, a public University and the Guardia Civil. Entomological evidence was required to estimate the PMI in order to settle the perimortem circumstances. Piophila megastigmata (Diptera: Piophilidae) is reported for the first time from a forensic case in Spain, enhancing the interest of its presence in corpses to estimate the PMI. The procedure followed for each scientific specialist is detailed and the possibilities and requirements of each discipline are discussed in detail. Taking into account that institutional interdisciplinary collaboration is not a rule in solving cases of forensic investigation in Spain, the relevance and need of such interdisciplinary collaboration in solving cases of forensic investigation is emphasized.

The optimal post-eclosion interval while estimating the post-mortem interval based on an empty puparium.

The puparium is the hardened exoskeleton of the last larval instar of a fly, inside which a prepupa, a pupa and a pharate adult fly successively develop. Empty puparia are frequently collected at death scenes, especially in cases with a long post mortem interval (PMI). Although we are not able to estimate the interval between the eclosion of an adult fly and the collection of an empty puparium (i.e. the post-eclosion interval (PEI)), empty puparia may still provide valuable evidence about the minimum PMI. However, because of the unknown PEI, it is impossible to determine the time when the fly emerged, and thus when the retrospective calculation of the minimum PMI should start. In this study, the estimation of PMI (or minimum PMI) for empty puparia of Protophormia terraenovae Rob.-Desv. (Calliphoridae) and Stearibia nigriceps Meig. (Piophilidae) was simulated, to gain insight into the changes in estimates, when different PEIs and different temperature conditions were assumed. The simulations showed that the PEI (in a range of 0-90 days) had no effect on the PMI (or minimum PMI) when the puparium was collected in winter or early spring (December-April). In late spring, summer, or autumn (May-November) the PMI (or minimum PMI) increased with the PEI. The increase in PMI was large in the summer months, and surprisingly small in the autumn months, frequently smaller than the PEI used in the estimation. The shortest PMI was always obtained with a PEI of 0, indicating that the true minimum PMI is always estimated using a PEI of 0. When the puparium was collected during spring, simulations indicated that oviposition had occurred in the previous year, while in summer the previous-year oviposition has been indicated by the simulations only when longer PEIs had been assumed. These findings should guide estimation of the PMI (or minimum PMI) based on an empty puparium.

Dietary Effects on the Development of Calliphora dubia and Chrysomya rufifacies (Diptera: Calliphoridae): Implications for Postmortem Interval.

Forensic entomology relies on insect development data generated within a laboratory setting in the estimation of minimum postmortem interval (mPMI). The methodologies used to produce these data vary considerably within the field and there is no accepted standard approach to laboratory rearing of forensically relevant species. A wide range of rearing media are used across published studies, including different species of animal and types of tissue (e.g., muscle and liver). Differing methodologies, particularly rearing diet, can introduce considerable variation into the baseline data upon which forensic estimates of the mPMI are calculated. Consequently, research establishing a widely available, standard and/or optimal, rearing medium for blow fly development for forensic application is desirable. This study examined dietary effects on the development of two forensically relevant blow fly species: Calliphora dubia Macquart, 1855, and Chrysomya rufifacies Macquart 1842 (Diptera: Calliphoridae). Larvae of both species were reared on pork liver, pork mince, pork loin, beef liver, beef mince, and guinea pig carcass under two constant temperature regimes (24 ± 1°C and 30 ± 1°C; 70 ± 10% humidity; 12-h/12-h photoperiod) to assess the influence of temperature on developmental response to diet. Fundamental developmental data pertaining to both species are reported. Developmental response to diet was species-specific and influenced by temperature with indication that the optimal temperature for C. dubia development is below 30°C. Pork mince was the most appropriate dietary standard of the rearing media investigated for the formulation of forensic development data for both species investigated.

Role of Chrysomya albiceps (Diptera: Calliphoridae) and Musca domestica (Diptera: Muscidae) Maggot Crop Contents in Identifying Unknown Cadavers.

Forensic entomology focuses on the analysis of insect larvae present at crime scenes to help identify unknown cadavers. Carrion-feeding maggots store food in a crop located at the anterior end of the gut. DNA recovered from the crop can be amplified, sequenced, and identified to determine the origin of the food. This information could help investigators to identify a missing victim if maggots are discovered at a crime scene in the absence of a corpse. Chrysomya albiceps (Wiedemann) and Musca domestica (Linnaeus) are primary forensic species. Little or no information on the identification of unknown cadavers using C. albiceps and M. domestica larvae is available, and we aimed to compare the effectiveness of using the crop contents of instars of C. albiceps and M. domestica larvae to identify corpses at different time intervals. Two hundred and forty larvae of both species were reared on rat and/or mouse liver, and DNA from crop contents was extracted after different time intervals from different instar larvae. DNA was amplified using specific primers that match the mitochondrial cytochrome oxidase subunit I (mt COI) gene of the food source. Results showed that food provided to larvae affected life span and the ability to be used to identify unknown cadavers. Chrysomya albiceps larvae proved more useful than M. domestica larvae. Moreover, crop contents of third instar larvae of both species fed for 24 h are more useful than contents from other time intervals in identifying unknown tissues. Results are promising and may help investigators to identify unknown/missing victims.

Development of Hydrotaea spinigera (Diptera: Muscidae) at Constant Temperatures and Its Significance for Estimating Postmortem Interval.

Hydrotaea spinigera Stein is a necrophagous species, widely distributed in Oriental and Australasian regions. Considering that the postfeeding larvae or puparia of this species can still be found in abundance at the advanced decomposition stage or even the skeleton stage of remains, it can serve as a good supplementary indicator for estimating the minimum postmortem interval (PMImin). This could also extend the range of PMImin when the primary colonizers are no longer associated with the corpse or have emerged as adults. This study investigated the development duration, accumulated degree hours, and larval body length changes of H. spinigera at seven constant temperatures ranging from 16 to 34°C, and established three development models for estimating PMImin, including isomorphen diagram, isomegalen diagram, and thermal summation model. At 16, 19, 22, 25, 28, 31, and 34°C, the development durations of H. spinigera from egg to adult stage were 1,412.6 ± 62.9, 867.4 ± 14.9, 657.1 ± 22.9, 532.3 ± 10.1, 418.8 ± 21.3, 379.8 ± 16.6, and 340.0 ± 20.3 h, respectively. The lower developmental threshold L0 was estimated as 10.50 ± 0.20°C, and the thermal summation constant K was 7,648.83 ± 146.74 degree hours. Using regression analysis, equations were obtained modeling the change of larval body length with time after hatching at different temperatures. This study provided basic data based on the growth and development of H. spinigera for the estimation of PMImin in forensic science.

Factors Affecting the Composition and Succession of Beetles in Exposed Pig Carcasses in Southern Brazil.

Coleoptera is one of the largest taxon among animals and exhibits diverse eating habits. When associated with decaying corpses, beetles can be of great value in estimating the postmortem interval. In order to consolidate a useful database for the forensic field, it is necessary to study the entomological fauna associated with the carcasses in different geographical regions since the diversity of insects varies according to the biogeoclimate zone. Thus, this study aimed to assess the influence of environmental and ecological factors on the composition and succession of beetles associated with pig carcasses exposed in southern Brazil. Collections were carried out during the hot/dry and cold/wet seasons. A total of 415 specimens belonging to 18 Coleoptera families were sampled. The highest total abundance (n = 329) and diversity (n = 44 taxa) were recorded in the cold/wet season, corresponding to approximately 80% of the total sampled from the two seasons. Dermestidae (26.7%) was the family most abundantly sampled. Regarding eating habits, in an increasing order of importance were necrophagous (43.3%), predator (31.6%), and omnivorous (0.05%). In the hot/dry season, there was no faunal succession. In the cold/wet season, the succession was more associated with differences in abundance than to the presence or absence of a specific taxon by decomposition stage. Considering all the factors analyzed in the current study, three species of beetles, Dermestes maculatus DeGeer (Dermestidae), Euspilotus azureus (Sahlberg, 1823) (Histeridae), and Oxelytrum discicolle Brullé, 1840 (Silphidae), could be identified as being of the greatest forensic relevance in this biogeoclimatic zone.

Developmental Biology of Forensically Important Beetle, Necrophila (Calosilpha) brunnicollis (Coleoptera: Silphidae).

The postmortem interval (PMI) estimation, in cases where the body was discovered in an advanced stage of decomposition, is predominantly based on entomological evidence. However, very few forensically important species are sufficiently known in detail to allow a practical application. One of them is the carrion beetle, Necrophila (Calosilpha) brunnicollis (Kraatz, 1877). Its development from egg to adulthood was studied under a range of ecologically relevant constant temperatures to find parameters of thermal summation models. Developmental sexual dimorphism and the presence of developmental rate isomorphy were investigated. Herein we present the lower developmental thresholds and sum of effective temperatures for all developmental stages of N. brunnicollis (egg, first-third larval instar, postfeeding stage, and pupae). We did not find any evidence of developmental sexual dimorphism nor was the presence of developmental rate isomorphy confirmed. Our results present the first thermal summation model of the East Asian carrion beetle that can be used for the PMI estimation.

Cuticular hydrocarbons as a tool for determining the age of Chrysomya rufifacies (Diptera: Calliphoridae) larvae.

Calliphoridae are one of the most important insect groups encountered as evidence collected from a crime scene. Age determination of the immature stages of these necrophagous flies is an important step toward estimating the time of colonization and inferring a minimum postmortem interval (PMImin ) in most instances. To determine if the cuticular hydrocarbons could be used to establish whether the development stages yield characteristics profiles, allowing for age estimation, hydrocarbons were extracted from 1st and 2nd, as well as feeding and post-feeding 3rd instar Chrysomya rufifacies, the hairy maggot blow fly. Extracted hydrocarbons were analyzed using gas chromatography coupled to mass spectrometry with the aim to investigate the changes in chemical profiles of each larval stage. A total of 23 compounds were identified with most of them being alkanes (65%) with carbon chain lengths of 9-33 carbons, alkenes (18%), and methyl-branched alkanes (17%). All the hydrocarbons except pentadecane (C15), hexadecane (C16), and nonacosane (C29) showed significant differences in their expression throughout larval development. For 1st instars, nonane was the most abundant (17% of the total hydrocarbons content) compound. Accounting for 11% and 10% of the cuticular hydrocarbons, tricosane and pentacosane, respectively, were the notable hydrocarbons in 2nd instars. For post-feeding 3rd instars, hentriacontane and tritriacontane were present with relative abundances 18% and 15%, respectively. On average, there was a shift from low to high molecular weight hydrocarbons as the larvae aged. These results indicate the change in hydrocarbons makeup as larvae age and could potentially be used to determine the age of immature C. rufifacies and hence aid in PMImin estimations. However, future research is needed to validate these results under natural conditions in the field.

Evaluation of Reference Genes and Age Estimation of Forensically Useful Aldrichina grahami (Diptera: Calliphoridae) During Intrapuparial Period.

The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (ß-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.

Contributions to the Estimation of the Postmortem Interval Through the Length and Body Weight of Two Indigenous Species of South America: Lucilia ochricornis (Diptera: Calliphoridae) and Lucilia purpurascens.

The early arrival and colonization of species belonging to the family Calliphoridae (Insecta: Diptera) on a corpse represent one of the most reliable means of estimating minimum postmortem interval (PMImin). However, information on the development and life cycles of some Argentine species in this family is not complete. The objective of this work was to contribute new information regarding the larval body size of neotropical species that allow, through the construction of forensic methods, the estimation of a more precise and specific PMImin. This work was conducted on laboratory cultures of larvae of Lucilia ochricornis (Wiedemann) and Lucilia purpurascens (Walker) using as average temperatures: 13.4, 15.1, 22.6, and 23.3°C, which represent the four seasons of the year for the province of Salta. With this information, we constructed isomegalen diagrams and growth models for the obtained variables of larval length and body weight. The mean values of length and body weight differ between both species, indicating that L. purpurascens exceeded L. ochricornis in both variables. In contrast, within each species the mean length and weight remained unchanged between culture temperatures for the three larval instars. Isomegalen diagrams can be used for the entire range of temperatures worked in the laboratory, but the body size entered is approximate. The growth models allow the use of point data but are specific for each culture temperature used.

Standardized Laboratory Methodology for the Evaluation of Foraging Strategies in Necrophilous Beetles: A Case Study of Necrophila (Calosilpha) brunnicollis (Coleoptera: Silphidae).

Precise data regarding feeding habits of necrobiont species are a key element of food web and evolutionary ecology. They can also be used to assess the utility and value of those species for forensic entomology, where obligatory necrophagous species in particular are considered good bioindicators of postmortem or preappearance interval. However, the feeding habits of many species are known only from anecdotal field observations, often reduced to vaguely defined categories-predatory, necrophagous, or omnivorous. To address this issue, we designed a simple, in vitro behavioral experiment allowing the quantification of food preferences. Next, we applied it on Necrophila (Calosilpha) brunnicollis (Kraatz, 1877), which is a common carrion beetle of East Asia with unresolved food preferences. The results suggest that this species is preferentially necrophagous, thus valuable for forensic research. Importantly, however, our experimental design allowed us to reveal that it also readily feeds on larvae of Diptera, although they compose a minor proportion of its diet. This methodology can be applied to other species, and it could provide evidence for future decision making in forensic research.

Development of Megaselia spiracularis (Diptera: Phoridae) at different constant temperatures.

Megaselia spiracularis Schmitz, 1938 (Diptera: Phoridae) is a pest that often appears in human living areas where it can spread pathogens. Besides, the species is of forensic value. Currently, studies focusing on the development of this species are limited. Understanding the developmental patterns of M. spiracularis, therefore, is important for controlling populations of this pest and for estimating the minimum postmortem interval (PMImin). Here, we studied the development of M. spiracularis exposed to seven constant temperatures ranging from 16 to 34 °C. The developmental durations, accumulated degree hours and larval body length changes were measured. Three kinds of development models that can be used to estimate the PMImin were established, including isomorphen diagram, isomegalen diagram and thermal summation model. The duration of M. spiracularis development at 16, 19, 22, 25, 28, 31 and 34 °C from egg to adult stage were 1131.1 ± 34.5, 807.3 ± 9.3, 529.6 ± 1.8, 367.0 ± 8.8, 302.4 ± 7.0, 250.0 ± 2.1 and 232.6 ± 1.9 h, respectively. The developmental threshold temperature and the thermal summation constant were estimated as 12.0 ± 0.5 °C and 4989.7 ± 308.9° hours, respectively. A general model represented by a logistic equation describing how larval body length will change with the time after hatching was fit to data. The present study provides basic developmental data of M. spiracularis, which can be used for achieving better control of this noxious insect as well as for estimation of its PMImin at different temperatures.

Development of Sarcophaga dux (diptera: Sarcophagidae) at constant temperatures and differential gene expression for age estimation of the pupae.

Sarcophaga dux (Diptera: Sarcophagidae) is a necrophagous flesh fly species with potential forensic value for estimating minimum postmortem interval (PMImin). The basic developmental data and precise age estimates of the pupae are significant for PMImin estimation in forensic investigations. In the present study, we investigated the development data of that species at seven constant temperatures varying from 16 °C to 34 °C, including body length changes of the larve, developmental duration and accumulated degree hours of the preadults. Several reference genes for relative quantification of the differentially expressed genes (DEGs) were firstly selected and evaluated in the pupae of different ages under different temperatures. The DEGs of the insects during the pupal period at different constant temperatures (34, 25 and 16 °C) were further analyzed for more precise age estimation. The results showed that the developmental durations of the preadults at 16, 19, 22, 25, 28, 31 and 34 °C were 1478.6 ± 18.3 h, 726.1 ± 15.8 h, 538.5 ± 0.9 h, 394.1 ± 9.5 h, 375.6 ± 10.8 h, 284.1 ± 7.3 h, and 252.5 ± 6.1 h, respectively. The developmental threshold temperature the flies was 12.27 ± 0.35 °C, and the thermal summation constant was 5341.71 ± 249.29° hours. The most reliable reference genes during the pupal period at different temperatures were found: GST1 and 18S rRNA for the 34 °C group, GST1 and RPL49 for 25 °C, and 18S rRNA and 28S rRNA for 16 °C. The four differential expression genes (Hsp60, A-alpha, ARP, and RPL8) have the potential to be used for more precise age estimation of pupal S. dux. This work provides important basic developmental data and a more precise age estimation method for pupal S. dux, and improves the value of this species for PMImin estimation in forensic investigations.

Age determination of the adult blow fly Lucilia sericata (Diptera: Calliphoridae) through quantitative pteridine fluorescence analysis.

Determination of a minimal postmortem interval via age estimation of necrophagous diptera has been restricted to the juvenile stages and the time until emergence of the adult fly, i.e. up until 2-6 weeks depending on species and temperature. Age estimation of adult flies could extend this period by adding the age of the fly to the time needed for complete development. In this context pteridines are promising metabolites, as they accumulate in the eyes of flies with increasing age. We studied adults of the blow fly Lucilia sericata at constant temperatures of 16 °C and 25 °C up to an age of 25 days and estimated their pteridine levels by fluorescence spectroscopy. Age was given in accumulated degree days (ADD) across temperatures. Additionally, a mock case was set up to test the applicability of the method. Pteridine increases logarithmically with increasing ADD, but after 70-80 ADD the increase slows down and the curve approaches a maximum. Sex had a significant impact (p < 4.09 × 10-6) on pteridine fluorescence level, while body-size and head-width did not. The mock case demonstrated that a slight overestimation of the real age (in ADD) only occurred in two out of 30 samples. Age determination of L. sericata on the basis of pteridine levels seems to be limited to an age of about 70 ADD, but depending on the ambient temperature this could cover an extra amount of time of about 5-7 days after completion of the metamorphosis.

Eye-background contrast as a quantitative marker for pupal age in a forensically important carrion beetle Necrodes littoralis L. (Silphidae).

Insect pupae sampled at a death scene may be used to estimate the post-mortem interval. The pupal age is however difficult to estimate, as there are no good quantitative markers for the age of a pupa. We present a novel method for pupal age estimation based on the quantification of contrast in intensity between the eyes of a pupa and the middle grey photography card as a standard background. The intensity is measured on a standardized scale from 0 (perfect black) to 255 (perfect white) using computer graphical software and pictures of the eye and the background taken with a stereomicroscope. Eye-background contrast is calculated by subtracting the average intensity of the eye from the average intensity of the background. The method was developed and validated using pupae of Necrodes littoralis (Linnaeus, 1758) (Coleoptera: Silphidae), one of the most abundant beetle species on human cadavers in Central Europe. To develop the model, pupae were reared in 17, 20 and 23 °C, with a total of 120 specimens. The method was validated by three raters, using in total 182 pupae reared in 15, 17, 20, 23 and 25 °C. We found a gradual increase in eye-background contrast with pupal age. Changes followed generalized logistic function, with almost perfect fit of the model. Using our method pupal age was estimated with the average error of 8.1 accumulated degree-days (ADD). The largest error was 27.8 ADD and 95% of age estimates had errors smaller than 20 ADD. While using the method, different raters attained similar accuracy. In conclusion, we have demonstrated that eye-background contrast is a good quantitative marker for the age of N. littoralis pupae. Contrast measurements gave accurate estimates for pupal age. Our method is thus proven to be a candidate for a reliable approach to age insect pupae in forensic entomology.