Improving accuracy of age estimates for insect evidence-calibration of physiological age at emergence (k) using insect size but without "k versus size" model.

Postmortem interval may be estimated based on the age of insect evidence collected on a death scene. Reference data that are used in such estimation frequently comprise thermal summation constant (i.e. k), which is equal to the insect age upon completion of immature development expressed in accumulated degree-days or degree-hours (ADD or ADH). Essentially, k is a central point of an insect group and it may poorly represent insect evidence that is near the limits of variation for the group. Accordingly, it was postulated to calibrate k for particular insect evidence and insect size and sex were found to be useful for this purpose in some of the species. However, the calibration is only possible by using the model that correlates k with insect size. Since very few such models were published, this lack of data limits the calibration of k in forensic casework. In this article, we develop a formula that is useful for the calibration of k without the use of "k versus size" model (and related datasets). The formula uses k from the general thermal summation model for a species (with its standard error), size range for the species (retrieved from entomology literature), and size measurements for particular insect evidence. The calibration of k with the formula was validated using the Creophilus maxillosus (Coleoptera: Staphylinidae) and Necrodes littoralis (Coleoptera: Silphidae) datasets. It was particularly useful while analyzing unusually small and large insects, in case of which the formula reduced the inaccuracy of k from the general model on average by ~25 ADD in C. maxillosus and ~40 ADD in N. littoralis. We discuss the limitations and prospects of the calibration protocol that employs the formula.

Competition, cooperation, and parental effects in larval aggregations formed on carrion by communally breeding beetles Necrodes littoralis (Staphylinidae: Silphinae).

Aggregations of juveniles are dominant forms of social life in some insect groups. Larval societies are shaped by competitive and cooperative interactions of the larvae, in parallel with parental effects. Colonies of necrophagous larvae are excellent systems to study these relationships. Necrodes littoralis (Staphylinidae: Silphinae), a carrion beetle that colonizes cadavers of large vertebrates, forms massive juvenile aggregations. By spreading over carrion anal and oral exudates, the beetles form the feeding matrix, in which the heat is produced and by which adults presumably affect the fitness of the larvae. We predict that exploitative competition shapes the behavior of N. littoralis larvae in their aggregations. However, cooperative interactions may also operate in these systems due mainly to the benefits of collective exodigestion. Moreover, indirect parental effects (i.e., formation of the feeding matrix) probably modulate larval interactions within the aggregations. By manipulating parental effects (present/absent) and larval density (0.02-1.9 larvae/g of meat), we found a strong negative group-size effect on fitness components of N. littoralis, in colonies with parental effects over almost the entire density range, and in colonies without parental effects for densities larger than 0.5 larva/g. This was accompanied by positive group-size effects in terms of development time (it shortened with larval density) and thermogenesis (it increased with larval density). A pronounced positive group-size effect on juvenile fitness was found only in colonies without parental effects and only in the low-density range. These results support the hypothesis that larval societies of N. littoralis are shaped by exploitation competition.

Field validation of post-mortem interval estimation based on insect development. Part 1: Accuracy gains from the laboratory rearing of insect evidence.

There is a consensus that forensic methods must be valid. The high quality of the method may be fully demonstrated only through validation. Unfortunately, there are very few experimental or casework validations of entomological methods for estimating post-mortem interval (PMI). Here, we present the first part of the results from the field validation of minimum PMI (PMImin) estimates based on insect development. From eight pig carcasses (24-46.4 kg) exposed in a forest habitat of Western Poland, one every one or two months, we collected insect evidence with standard entomological techniques. Using weather station temperatures and the thermal summation method, PMImin was estimated based on insect life stages that were reared under controlled laboratory conditions. Through rearing an insect until the next developmental landmark (or until eclosion), its age at the time of collection (and eventually PMImin) may be estimated by subtracting thermal accumulation in the laboratory from the corresponding thermal constant. We hypothesized that rearing insect evidence significantly improves the accuracy of PMImin compared to the estimation based on non-reared insect evidence. The results clearly supported this hypothesis. However, the accuracy significantly increased only in the case of these insects that developed normally during rearing. When their development in the laboratory was prolonged, PMImin was significantly less accurate. For the normal development sample the accuracy improved in all species and life stages. The largest accuracy gains from rearing were recorded for Calliphora vomitoria (Diptera: Calliphoridae) and Stearibia nigriceps (Diptera: Piophilidae). Moreover, when puparia or third instar larvae were reared, gains were larger than in the case of earlier life stages. In conclusion, this study demonstrated that the method of rearing insect evidence to improve the accuracy of PMImin is valid. However, it needs to be used with caution, since substantial part of the evidence may die or slow down their development in the laboratory, which violates assumptions of the method.

Calibrating insect age at eclosion by size in a gregarious carrion beetle Thanatophilus sinuatus (Staphylinidae: Silphinae).

Recent discoveries have shown that the physiological age at eclosion of forensically useful beetles differs between males and females and between beetles of various sizes. Accordingly, it was postulated that the size and sex of the beetles at eclosion may be used to calibrate their age, which may improve the accuracy of age (and post-mortem interval) estimates in forensic entomology. In this study, we derived thermal summation models for the eclosion for the Central European population of carrion beetles Thanatophilus sinuatus (Fabricius, 1775), (Staphylinidae: Silphinae), and tested the usefulness of sex and size for the calibration of beetle age at eclosion. Although in previous developmental studies, the beetles were reared individually, we reared them in larval aggregations, since in natural conditions T. sinuatus beetles are gregarious. Weak (r2 between 5% and 13%) negative correlations were observed between the size and age of T. sinuatus males or females at eclosion, demonstrating that calibration of age by beetle size and sex may bring only minimal benefits regarding the accuracy of age estimation in this species. However, it may still be worthwhile in the case of extremely large or small beetles. Moreover, the total development times recorded in this study were much shorter than in the previous T. sinuatus study, at 14°C by about 15 days and at 26°C by about 2 days. These differences emphasise the importance of gregariousness for the development of carrion beetles, and at the same time highlight the need for the ecologically-relevant protocols of development studies in forensic entomology.

Initial laboratory validation of temperature development models for Necrodes littoralis L. (Staphylinidae: Silphinae).

Development models of necrophagous insects are applied in forensic entomology for post-mortem interval estimation. Such estimates may be used as scientific evidence in legal investigations. For this reason, it is important that the models are valid and that the expert witness is aware of their limitations. Necrodes littoralis L. (Staphylinidae: Silphinae) is a necrophagous beetle species that frequently colonizes human cadavers. Temperature models of development for the Central European population of these beetles were recently published. In this article, we present results of the laboratory validation study for these models. Errors of beetle age estimation differed significantly between the models. Thermal summation models yielded the most accurate estimates, and the isomegalen diagram least accurate estimates. Errors of the beetle age estimation varied across beetle developmental stages and rearing temperatures. In general, most development models of N. littoralis were satisfactorily accurate in estimating beetle age under laboratory conditions; therefore, the study provides initial evidence to support their validity in forensic cases.

Temperature models of development for Necrodes littoralis L. (Coleoptera: Silphidae), a carrion beetle of forensic importance in the Palearctic region.

Analysis of insects can provide evidence in death cases, for example, by answering the question about the time of death. Apart from flies, beetles are the second most useful insect group in forensic entomology. To elucidate the time of death based on insect evidence, developmental models of a given species are necessary. In this study, we developed such models for Necrodes littoralis, a necrophagous beetle, which is common in the Palearctic region and has great potential in forensic entomology. We monitored the development at 10 constant temperatures (14-30 °C). Larvae were reared in aggregations. Thermal summation models, isomorphen and isomegalen diagrams and growth curves were derived using the data. Depending on the temperature, development lasted between about 23 and 89 days. Mortality was high at the extremes of the temperature range. The thermal summation constant for the total development was 434.7 ± 28.86 accumulated degree-days above a developmental threshold of 9.04 ± 0.55 °C. This is the first comprehensive dataset on the development of N. littoralis. Implications for its use in forensic casework are discussed.

Competition of insect decomposers over large vertebrate carrion: Necrodes beetles (Silphidae) vs. blow flies (Calliphoridae).

Large carrion is inhabited by highly variable and interactive communities of insects. Positive interactions in carrion insect communities have been recently the focus in carrion ecology. In contrast, competition between carrion insects is rather undervalued. Here we provide evidence that blow flies (Calliphoridae) and Necrodes beetles (Silphidae), dominant decomposers of large carcasses in terrestrial habitats, compete over carrion. By reanalyzing the results from 90 pig carcasses, we demonstrated that the contribution of the flies and the beetles to the decay was negatively related. The greater part of the large carrion pool was monopolized by blow flies, whereas Necrodes beetles abundantly colonized carcasses, on which blow flies were less effective as decomposers. In behavioral assays, we found that adult beetles killed 4 times more frequently feeding than postfeeding third instar larvae of the flies, with the large decrease in the killing frequency after the larvae reached the age of early third instar. Therefore, adult Necrodes beetles preferentially killed the larvae that were before or in their peak feeding. The study provides evidence that the interaction between blow flies and Necrodes beetles is a combination of indirect exploitative effects of the flies and direct interference effects of the beetles (the mixed competition).

Erratum to: Competition of insect decomposers over large vertebrate carrion: Necrodes beetles (Silphidae) vs. blow flies (Calliphoridae).

[This corrects the article DOI: 10.1093/cz/zoab100.].

Insect rearing protocols in forensic entomology: Benefits from collective rearing of larvae in a carrion beetle Necrodes littoralis L. (Silphidae).

Forensic entomologists frequently use a developmental method to estimate a post-mortem interval (PMI). Such estimates are based usually on the blow fly larvae or puparia. Data on their development is obtained by rearing them in colonies. In the case of beetles, which can be also useful for PMI estimation, development data is frequently collected by rearing them individually. However, some carrion beetles are gregarious, for instance, Necrodes littoralis (Linnaeus, 1758) (Silphidae). We compared mortality, rate of development and body size of emerged adult beetles reared individually and in aggregations. Mortality was much higher for beetles reared individually, particularly at low temperatures. The rearing protocol affected the time of immature development and the size of adult insects. Individually reared specimens developed much longer at 16°C, whereas at 20°C and 26°C development times of individually reared beetles were slightly shorter. Significant differences in the body size were observed only at 16°C; beetles that developed in aggregations were larger at this temperature. These findings demonstrate that aggregating is particularly beneficial for larvae of N. littoralis at low temperatures, where it largely reduces mortality and facilitates growth. Moreover, these results indicate that in forensic entomology the protocol of individual rearing is unsuitable for gregarious beetles, as it produces reference developmental data of low quality.

Post-Mortem Interval Estimation Based on Insect Evidence: Current Challenges.

During death investigations insects are used mostly to estimate the post-mortem interval (PMI). These estimates are only as good as they are close to the true PMI. Therefore, the major challenge for forensic entomology is to reduce the estimation inaccuracy. Here, I review literature in this field to identify research areas that may contribute to the increase in the accuracy of PMI estimation. I conclude that research on the development and succession of carrion insects, thermogenesis in aggregations of their larvae and error rates of the PMI estimation protocols should be prioritized. Challenges of educational and promotional nature are discussed as well, particularly in relation to the collection of insect evidence.

Heat production in a feeding matrix formed on carrion by communally breeding beetles.

Insects regulate their body temperature mostly behaviourally, by changing posture or microhabitat. Usually they use heat that is already present in the environment. Sometimes, however, they may manipulate the environment to affect, focus or benefit from thermogenesis. Carrion beetles create a feeding matrix by applying to cadaver surface anal or oral exudates. We tested the hypothesis that the matrix, which is formed on carrion by communally breeding beetle Necrodes littoralis L. (Silphidae), produces heat that enhances insect fitness. Using thermal imaging we demonstrate that heat produced in the matrix formed on meat by adult or larval beetles is larger than in meat decomposing without insects. Larval beetles regularly warmed up in the matrix. Moreover, by comparing matrix temperature and larval fitness in colonies with and without preparation of meat by adult beetles, we provide evidence that formation of the matrix by adult beetles has deferred thermal effects for larval microhabitat. We found an increase in heat production of the matrix and a decrease in development time and mortality of larvae after adult beetles applied their exudates on meat in the pre-larval phase. Our findings indicate that spreading of exudates over carrion by Necrodes larvae, apart from other likely functions (e.g. digesting carrion or promoting growth of beneficial microbes), facilitates thermoregulation. In case of adult beetles, this behaviour brings distinct thermal benefits for their offspring and therefore may be viewed as a new form of indirect parental care with an important thermal component.

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.

Estimation of physiological age at emergence based on traits of the forensically useful adult carrion beetle Necrodes littoralis L. (Silphidae).

The main entomological method for post-mortem interval involves estimation of age for immature insects found on a cadaver. Forensic entomologists frequently use the thermal summation value for the total immature development (K), which is a measure of physiological time needed to complete development of a species (age at emergence). K is highly variable within the species. Its true value for an adult insect may be estimated based on insect traits such as size and sex at maturity. Here, we have tested, if size and sex of adult beetles of Necrodes littoralis may be useful for the estimation of the true K. Necrodes littoralis is a Palearctic carrion beetle that frequently colonizes human cadavers in forest and agricultural environments. General and sex-specific thermal summation models for the emergence and models for the relationship between size of adult beetles and their age at emergence were developed for N. littoralis. The models were subsequently tested in the validation study. The general K for N. littoralis was about 469 (+/-25 SE) accumulated degree-days above the developmental threshold of about 8.5 (+/-0.45 SE) °C. Thermal summation parameters of the sex-specific models revealed minor differences compared to the general model. A true K was negatively related to the beetle size. Methods for the estimation of K represented its true value with different accuracy. The highest accuracy was obtained when K was estimated using beetle weight as a predictor variable and the sex-specific models for the relationship between K and size, although sex contributed slightly to this improvement. Using this method the estimated K represented the true K with the error of 6.3%, while the error for K from the general thermal summation model was about 9.7%. In conclusion, the findings demonstrate that physiological age at emergence of N. littoralis may be accurately predicted based on the adult beetle size. Necrodes littoralis is a second beetle species in which the age at maturity was more accurately represented by size-based estimates of K than K from the general thermal summation model. Therefore, we encourage testing the relationship between K and size in all insect species that are used in forensic entomology; particularly blow flies and flesh flies.

Correction to: Development and validation of forensically useful growth models for central European population of Creophilus maxillosus L. (Coleoptera: Staphylinidae).

This article was published with an error in Figs. 5 and 6. The figures have multiple mistakes with lines extending beyond the chart and lack of grid lines in some places.

Development and validation of forensically useful growth models for Central European population of Creophilus maxillosus L. (Coleoptera: Staphylinidae).

The hairy rove beetle, Creophilus maxillosus (Linnaeus) (Staphylinidae), is recognized for its use in forensic entomology. However, insufficient developmental data exist for the Central European population of this species. Accordingly, we studied the development of C. maxillosus at ten constant temperatures (10-32.5 °C). Based on these results, linear and nonlinear developmental models were created and validated. We also studied the effect of different homogenous diets (third-instar larvae or puparia of Calliphora sp. Robineau-Desvoidy or Lucilia sp. Robineau-Desvoidy (Diptera: Calliphoridae) or mix of first- and second-instar larvae of Necrodes littoralis (Linnaeus) (Coleoptera: Silphidae)) on the development and mortality of C. maxillosus. Average total development times ranged between 122.21 days at 15 °C and 22.18 days at 30 °C. Beetles reached the adult stage in seven out of ten temperatures (15-30 °C). No beetles reached the adult stage when fed with larvae of N. littoralis; their development times at first and second larval stage were also significantly longer than in other food conditions. When C. maxillosus larvae were fed with blowfly larvae, the highest mortality was observed at the pupal stage, as compared when they were fed with blowfly puparia-at the first larval stage. While validating thermal summation models, the highest age estimation errors were found for beetles bred at 10 and 12.5 °C (between 21 and 43% for all developmental events). Age estimation errors were on average higher for pupation and eclosion than hatching and first and second ecdyses. While validating the models with specimens fed with different diets, the highest errors were recorded for beetles fed with N. littoralis larvae (22% for the first ecdysis and 33% for the second ecdysis) and Lucilia sp. puparia (32% for pupation and 22% for eclosion). Implications for C. maxillosus use in forensic entomology are discussed.

Pigs vs people: the use of pigs as analogues for humans in forensic entomology and taphonomy research.

Most studies of decomposition in forensic entomology and taphonomy have used non-human cadavers. Following the recommendation of using domestic pig cadavers as analogues for humans in forensic entomology in the 1980s, pigs became the most frequently used model cadavers in forensic sciences. They have shaped our understanding of how large vertebrate cadavers decompose in, for example, various environments, seasons and after various ante- or postmortem cadaver modifications. They have also been used to demonstrate the feasibility of several new or well-established forensic techniques. The advent of outdoor human taphonomy facilities enabled experimental comparisons of decomposition between pig and human cadavers. Recent comparisons challenged the pig-as-analogue claim in entomology and taphonomy research. In this review, we discuss in a broad methodological context the advantages and disadvantages of pig and human cadavers for forensic research and rebut the critique of pigs as analogues for humans. We conclude that experiments using human cadaver analogues (i.e. pig carcasses) are easier to replicate and more practical for controlling confounding factors than studies based solely on humans and, therefore, are likely to remain our primary epistemic source of forensic knowledge for the immediate future. We supplement these considerations with new guidelines for model cadaver choice in forensic science research.

Post-mortem interval estimation based on insect evidence in a quasi-indoor habitat.

Insects collected on indoor cadavers are frequently used for post-mortem interval (PMI) estimation. Buildings encountered during crime investigations vary according to temperatures inside, the extent of insect access restriction or sanitary conditions. This article reports the PMI oriented analyses of insect evidence sampled from the human cadaver in the atypical indoor habitat. The body was found in the uninhabited house, on the floor covered with rubbish, in the room with no doors and windows. Thermal conditions in the room were less variable than in the local weather station, however still much more variable compared to the typical indoor habitat, indicating the need for retrospective correction of temperature records from the station. Cadaver entomofauna was surprisingly diverse and abundant. We recorded several taxa usually not occurring on indoor cadavers, e.g. immature stages of Necrodes littoralis (Coleoptera: Silphidae) or Stearibia nigriceps (Diptera: Piophilidae). PMI was based on the age and the pre-appearance interval estimated for live puparium of S. nigriceps, giving the total interval of 37 (±7.4) days plus 4-20 days resulting from the absence of first colonizing specimens of the species. This estimate was corroborated with the age estimate for empty puparia of Sarcophaga argyrostoma (Diptera: Sarcophagidae) with traces of Nasonia sp. (Hymenoptera: Pteromalidae) eclosion. Other insects indicated shorter but consistent PMI. Difficulties and limitations of insect-based PMI estimations in unusual indoor habitats are discussed.

Size at emergence improves accuracy of age estimates in forensically-useful beetle Creophilus maxillosus L. (Staphylinidae).

Insects colonizing human or animal cadavers may be used to estimate post-mortem interval (PMI) usually by aging larvae or pupae sampled on a crime scene. The accuracy of insect age estimates in a forensic context is reduced by large intraspecific variation in insect development time. Here we test the concept that insect size at emergence may be used to predict insect physiological age and accordingly to improve the accuracy of age estimates in forensic entomology. Using results of laboratory study on development of forensically-useful beetle Creophilus maxillosus (Linnaeus, 1758) (Staphylinidae) we demonstrate that its physiological age at emergence [i.e. thermal summation value (K) needed for emergence] fall with an increase of beetle size. In the validation study it was found that K estimated based on the adult insect size was significantly closer to the true K as compared to K from the general thermal summation model. Using beetle length at emergence as a predictor variable and male or female specific model regressing K against beetle length gave the most accurate predictions of age. These results demonstrate that size of C. maxillosus at emergence improves accuracy of age estimates in a forensic context.

Sex-specific developmental models for Creophilus maxillosus (L.) (Coleoptera: Staphylinidae): searching for larger accuracy of insect age estimates.

Differences in size between males and females, called the sexual size dimorphism, are common in insects. These differences may be followed by differences in the duration of development. Accordingly, it is believed that insect sex may be used to increase the accuracy of insect age estimates in forensic entomology. Here, the sex-specific differences in the development of Creophilus maxillosus were studied at seven constant temperatures. We have also created separate developmental models for males and females of C. maxillosus and tested them in a validation study to answer a question whether sex-specific developmental models improve the accuracy of insect age estimates. Results demonstrate that males of C. maxillosus developed significantly longer than females. The sex-specific and general models for the total immature development had the same optimal temperature range and similar developmental threshold but different thermal constant K, which was the largest in the case of the male-specific model and the smallest in the case of the female-specific model. Despite these differences, validation study revealed just minimal and statistically insignificant differences in the accuracy of age estimates using sex-specific and general thermal summation models. This finding indicates that in spite of statistically significant differences in the duration of immature development between females and males of C. maxillosus, there is no increase in the accuracy of insect age estimates while using the sex-specific thermal summation models compared to the general model. Accordingly, this study does not support the use of sex-specific developmental data for the estimation of insect age in forensic entomology.

Blowfly puparia in a hermetic container: survival under decreasing oxygen conditions.

Despite widely accepted standards for sampling and preservation of insect evidence, unrepresentative samples or improperly preserved evidence are encountered frequently in forensic investigations. Here, we report the results of laboratory studies on the survival of Lucilia sericata and Calliphora vomitoria (Diptera: Calliphoridae) intra-puparial forms in hermetic containers, which were stimulated by a recent case. It is demonstrated that the survival of blowfly intra-puparial forms inside airtight containers is dependent on container volume, number of puparia inside, and their age. The survival in both species was found to increase with an increase in the volume of air per 1 mg of puparium per day of development in a hermetic container. Below 0.05 ml of air, no insect survived, and above 0.2 ml of air per 1 mg of puparium per day, survival reached its maximum. These results suggest that blowflies reveal a single, general pattern of survival under decreasing oxygen conditions and that this pattern is a product of number of developing insects, their age and the initial amount of available air. Implications for forensic entomology are discussed.