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.

All insects matter: a review of 160 entomology cases from 1993 to 2007 in Switzerland-part I (Diptera).

Necrophagous Diptera are the most important group of insects used for the purposes of forensic entomology. While the most utilized fly family in this context is the family Calliphoridae, there are several other families that can be of great importance during real-case investigations. This article analyzes the necrophagous flies of all families recorded from 160 real cases in Switzerland between 1993 and 2007. A total of 56 species belonging to 16 families was identified with Calliphoridae being the most dominant family (90.63% of all cases), followed by Muscidae (26.25%), Sarcophagidae (19.38%), Phoridae (14.38%), and Fanniidae (12.50%). For specimens that were difficult to identify morphologically, a new PCR primer has been specifically designed for the amplification of a short, informative COI barcode in degraded museum samples of forensically important Diptera taxa. The richest family in terms of species was the family Muscidae with 16 species. Fannia fuscula (Fallen) and Fannia monilis (Haliday) were recorded from human cadavers for the first time. The study highlights the importance of different fly families in forensic investigation, enhancing our comprehension of their prevalence and dispersion in real cases in Central Europe. The results pave the way for additional exploration, especially regarding the involvement of less frequently observed species in forensic entomology.

How to store a beetle larva? Comparing temporal effects of common fluid preservation methods on color, shape, and DNA quality.

Proper fixing and long-term preservation of entomological evidence are essential in collections and research and crucial in applied fields such as forensic entomology. Incorrectly stored samples may lose important morphological features over time, rendering molecular analyses exceedingly difficult. The most effective method for preserving soft samples such as larvae is fluid preservation. It uses a combination of a wide range of fixatives and storage fluids. However, very little comparative work has been done to determine the effects of long-term storage on sample quality in terms of color, shape, and DNA stability. Moreover, the current golden standard in forensic entomology has been tailored for age estimation of larvae of Diptera, which differ from larvae of Coleoptera in morphology and subsequently in applied methods. We compared the effects of combinations of 6 commonly used fixatives and 6 commonly used storage fluids on midsized larvae of the forensically important beetle, Necrodes littoralis (Linnaeus, 1758), in terms of color, shape, and suitability for DNA analyses over a 2-yr period. We were looking for combinations that can preserve specimens in a satisfactory state, can be used on a regular basis, do not require advanced protection or skills of the personnel, and are not toxic or too harmful to the environment. We found not only several methods that scored significantly better in the tested parameters compared with the golden standard but also several common methods that should be avoided. The effects of agents on each tested category are discussed in detail.

Integrative taxonomy and species distribution models of the genus Diamesus Hope, 1840 (Coleoptera: Staphylinidae: Silphinae).

Integrative taxonomy of Diamesus Hope, 1840 (Coleoptera: Silphinae) is presented. Adults of D. bimaculatus Portevin, 1914 (endemic to Taiwan) and D. osculans (Vigors, 1825) (widely distributed from northern India to Australia) are redescribed, keyed and figured, including characters of the male and female genitalia of both species. Variation in elytral maculation in D. osculans is discussed and illustrated. The absence of diagnostic differences of D. osculans var. reductus Pic, 1917 from D. osculans is discussed, and the former name is confirmed as a junior subjective synonym of D. osculans. Types of all three names available were studied; a lectotype and paralectotypes are designated for the name D. osculans var. bimaculatus Portevin, 1914. Molecular phylogenetic analysis confirms the genus Diamesus is sister group to the genus Necrodes Leach, 1815, and D. osculans and D. bimaculatus are two, well supported clades. Detailed data on the distribution of D. bimaculatus and D. osculans are presented and mapped. Species distribution models for both species were created and interpreted. Diamesus osculans is reported for the first time from India: Uttarakhand, China: Anhui, Hainan, Hunan, Jiangxi, Shaanxi and Zhejiang Provinces, and Australia: Victoria; it is also recently confirmed from Taiwan, being sympatric in distribution there with D. bimaculatus. Available data on the ecology and seasonality of both species of Diamesus are also discussed.

Diet Impact on the Development and Survival of Oiceoptoma thoracicum (Coleoptera: Silphidae).

We assessed the influence of diet on the development and survival in the immature stages of the necrophagous beetle Oiceoptoma thoracicum (Linnaeus, 1758). The species is frequently observed on large cadavers, including humans, and thus can be of potential forensic relevance. We compared multiple meat tissues from three animal sources, and detected the optimal diet for rearing the species for further entomological and forensic objectives. We reared 203 individuals to adulthood at the constant temperature of 20°C. Our results represent the first robust data set of the development time for this species. They further confirmed a significant relationship between survival and the type of diet, as the highest survival rates were detected in larvae fed with both pork liver and pork muscle.

Spatio-temporal distribution and habitat preference of necrophagous Calliphoridae based on 160 real cases from Switzerland.

Necrophagous blowflies (Diptera: Calliphoridae) are of great importance particularly during investigations of suspicious deaths. Many studies have analyzed the distribution of blowflies based on pig experiments and baited trapping; however, data from real case scenarios are rarely used. In this article, the distribution of blowflies found during investigations of 160 real cases during 1993-2007 in Switzerland is evaluated based on habitat, altitude, and season. Ten species of blowflies were present in 145 out of the 160 cases. The most common species was Calliphora vicina, which occurs throughout the year and was present in 69 % of all cases. Lucilia sericata, Calliphora vomitoria, and L. caesar were identified among the rest of the flies as species of great forensic importance mainly due to their distributional patterns. After a comparison with a similar dataset from Frankfurt, Germany, some surprising differences were determined and discussed. The biggest discrepancies between our dataset and the German dataset were in the occurrences of L. sericata (30 % vs. 86 %, respectively), Phormia regina (5 % vs. 43 %), and L. ampullacea (1 % vs. 45 %). The life-history strategies and intraspecific behavioral variability of blowflies remain understudied, although they can be essential for an unbiased approach during a death investigation. Further research and comparison of occurrence patterns across the area of distribution of blowflies are therefore needed and recommended.

Three-dimensional liquid chromatography with parallel second dimensions and quadruple parallel mass spectrometry for adult/infant formula analysis.

Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the 1D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.

Description of larval morphology and phylogenetic relationships of Heterotemna tenuicornis (Silphidae).

Providing clear and detailed morphological descriptions of endemic species in limited areas enables new knowledge of their biology and ecology to be obtained through citizen science. This information can be further used for their protection. Our study presents the first morphological description of the larvae of all three instars of Heterotemna tenuicornis (Brullé, 1836), an endemic species of the Canary Islands that, together with H. britoi García & Pérez, 1996 and H. figurata (Brullé, 1839), belongs to the peculiar genus Heterotemna Wollaston, 1864. Furthermore, we present the first sequences of two mitochondrial genes (COI, 16S) obtained from larval specimens, and cross reference them with sequences from an adult specimen. Phylogenetic analysis of molecular data placed the genus Heterotemna within the genus Silpha Linnaeus, 1758, suggesting paraphyly of Silpha. In our study, we underline the importance of using a combination of morphological description and molecular data, that can be used for barcoding developmental stages which could not otherwise be definitely associated.

Influence of Diet on Development and Survival of Thanatophilus rugosus (Coleoptera: Silphidae).

This study examined the effects of various diets on the development time and survival of the carrion beetle Thanatophilus rugosus (Fabricius, 1775). We attempted to find the best diet for rearing the species in laboratory conditions for entomological research and forensic purposes, and to further understand its feeding habits. The larval stages were monitored while feeding on three types of meat under a constant temperature. We discovered that the shortest development time in total was achieved in larvae fed with pork liver, followed closely by pork muscle. The longest development and lowest survival rates were observed when introducing a chicken diet. We were also able to identify specific stages during which the diet significantly affected the development.

Benefits and Pitfalls of HPLC Coupled to Diode-Array, Charged Aerosol, and Coulometric Detections: Effect of Detection on Screening of Bioactive Compounds in Apples.

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.

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.

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.

Developmental Models of the Forensically Important Carrion Beetle, Thanatophilus sinuatus (Coleoptera: Silphidae).

Carrion beetles of genus Thanatophilus (Leach, 1815) are an important group of necrophagous insects, with great potential for forensic entomology in temperate zones of Africa, America, Asia, and Europe. Developmental models for majority of Thanatophilus species remain unknown. In this study, we will provide new thermal summation models for all the developmental stages of Thanatophilus sinuatus (Fabricius, 1775), one of the most abundant and widespread species of the genus. The beetles were bred at seven different constant temperatures, and developmental time was measured for each developmental stage (egg, three larval instars, postfeeding stage, and pupa). Temperature-sex influence was tested, and thermal summation constants were calculated to be used for postmortem interval estimation during criminal investigations.

Supercritical fluid chromatography in chiral separations: Evaluation of equivalency of polysaccharide stationary phases.

Nine different chiral columns based on covalently immobilized or coated tris(3,5-dimethylphenylcarbamate) cellulose and amylose have been explored. We evaluated their respective enantioselective potential including the enantioseparation and qualitative characteristics of peaks. The generic screening conditions were using gradient elution from 5 to 40% organic modifier/CO2 during 3 min with about 40 enantiomer pairs. Primary screening was carried out using ten different mobile phases varying in type of additives while using one representative amylose- and one cellulose-based column. The complete evaluation of all nine columns was then carried out using three best performing organic modifiers: (1) methanol + 0.1% trifluoroacetic acid + 0.1% diethylamine, (2) isopropanol + 0.1% trifluoroacetic acid + 0.1% diethylamine, and (3) methanol + 0.1% ammonium hydroxide. Equivalency of different columns with the same chiral selector was not confirmed. Columns with the same stationary phase but different supports or manufacturing methods displayed differences in enantioselectivity and general performance. The similarity corresponded to 62 and 63% for the three cellulose-coated columns taking CEL1 as the reference. The similarity was 67% for the pair of amylose-based coated columns. For immobilized columns, the similarity was 69 and 59% for celluloses and amyloses pairs, respectively. The best performing column based on success rate of enantioseparation was Chiralcel OD-3 when using methanol + 0.1% trifluoroacetic acid and 0.1% diethylamine combined additive.

The uptake, effects and biotransformation of monepantel in meadow plants used as a livestock feed.

Drugs are potentially dangerous environmental contaminants, as they are designed to have biological effects at low concentrations. Monepantel (MOP), an amino-acetonitrile derivative, is frequently used veterinary anthelmintics, but information about MOP environmental circulation and impact is almost non-existent. We studied the phytotoxicity, uptake and biotransformation of MOP in two fodder plants, Plantago lanceolata and Medicago sativa. The seeds and whole plant regenerants were cultivated with MOP. The plant roots and the leaves were collected after 1, 2, 3, 4, 5 and 6 weeks of cultivation. The lengths of roots and proline concentrations in the roots and leaves were measured to evaluate MOP phytotoxicity. The UHPLC-MS/MS technique with a Q-TOF mass analyser was used for the identification and semi-quantification of MOP and its metabolites. Our results showed no phytotoxicity of MOP. However, both plants were able to uptake, transport and metabolize MOP. Comparing both plants, the uptake of MOP was much more extensive in Medicago sativa (almost 10-times) than in Plantago lanceolate. Moreover, 9 various metabolites of MOP were detected in Medicago sativa, while only 7 MOP metabolites were found in Plantago lanceolata. Based on metabolites structures, scheme of the metabolic pathways of MOP in both plants was proposed. MOP and its main metabolite (MOP sulfone), both anthelmintically active, were present not only in roots but also in leaves that can be consumed by animals. This indicates the potential for undesirable circulation of MOP in the environment, which could lead to many pharmacological and toxicological consequences.

Description of immature stages of Thanatophilus sinuatus (Coleoptera: Silphidae).

Necrophagous beetles of genus Thanatophilus are well recognized as a group of beetles with a high potential utility in forensic entomology. They can be used to estimate postmortem interval (PMI) or validate the value for other groups of insects commonly encountered on human remains, like blowflies (Calliphoridae). However, reliable tools for instar and species identification of their larvae are needed as such information is crucial for allowing accurate PMI estimate. One of the most common species of the genus Thanatophilus in Europe is Thanatophilus sinuatus. This species occurs frequently on human remains and its larvae feed on decaying tissues throughout their development. Therefore, the larvae could become useful bioindicators for forensic entomology, although their current description does not allow reliable instar or species identification. Our goal was to provide morphological characters for species and instar identification of all larval stages of T. sinuatus. The larvae were obtained from laboratory rearing under controlled conditions (20 °C and 16:8 h of light/dark period). Qualitative and quantitative morphological instar and species-specific characters are described and illustrated. Additionally, we report observations of biological and developmental lengths for all stages of the species. We also compared these morphological characters with recent description of T. rugosus and provided an identification key of these two similar and often co-occurring species. We noticed that some characters for instar identification were shared between T. sinuatus and T. rugosus and were confirmed by comparison with larvae of T. dentigerus that they can be applied to other species of the genus.

Chroma+, a new automontage method of image background selection for Insects and other structurally complex objects.

Obtaining taxonomic-grade images is a vital part of probably every present-day morphological study of insects, even though the task itself is perceived as a "necessary evil" due to high investment of both time and effort to produce representable images. Cleaning the background and making it appear as a solid color of known properties is probably one of the most time-demanding tasks. Several techniques have been developed to reduce the time requirement; the most convenient and cost-effective one presumably being the chroma isolation. This method uses a green background that can be isolated and conveniently replaced with another picture or solid color, as used in the film industry. However, the main drawback of this technique is spilling of color onto the object, which is unavoidable and can be corrected only by sacrificing the true color of the object to some extent. Our improved Chroma+ method is based on classical chroma isolation workflow and helps to overcome this problem by taking an additional image of the object with a neutral color background and applying a selection obtained from the chroma-isolated picture on it. This technique is, in terms of the resulting image quality, superior to classical chroma isolation, while the time difference between these two methods is negligible. Furthermore, it does not require any additional equipment (hardware or software), thus being accessible to both employed taxonomists, low budget laboratories, and enthusiasts.

Revisited larval morphology of Thanatophilus rugosus (Coleoptera: Silphidae).

Determination of insect species and their instars, occurring on human remains, is important information that allows us to use insects for estimation of postmortem interval and detect possible manipulation with the body. However, larvae of many common species can be identified only by molecular methods, which is not always possible. The instar determination is even more challenging, and qualitative characters that would allow a more precise identification are mostly unknown. Thanatophilus rugosus (Linnaeus, 1758) is a common necrophagous beetle in the whole Palaearctic region from Europe to Japan. The species is often encountered on corpses of large vertebrates including humans, and its potential to become a useful bioindicator for forensic entomology is therefore high. Adults can be easily distinguished from other species; however, larvae were never thoroughly described to allow species and instar identification. The aim of this study was to provide reliable morphological characters that would allow species and instar identification of T. rugosus larvae. The material for morphological study was obtained from rearing under controlled conditions (20 °C and 12:12 h of light/dark period), and specimens that were not studied morphologically were allowed to complete their development. Quantitative and qualitative morphological characters for instar and species identification are described and illustrated. Additionally, we report observations of biology and developmental length for all stages of the species.

Effects of Bait Presence and Type of Preservative Fluid on Ground and Carrion Beetle Samples Collected by Pitfall Trapping.

Pitfall trapping is a sampling technique frequently used by entomologists around the world. However, there exist sampling biases linked to particular trapping designs, which require investigation. In this study, we compared the effects of the type of preservative fluid (propylene glycol or formaldehyde) and the presence of fish bait in pitfall traps on the number of specimens (individuals) collected, the species richness, and the species composition of carabid (Coleoptera: Carabidae) and silphid (Coleoptera: Silphidae) beetle assemblages. Traps containing propylene glycol collected a substantially higher number of individuals of both taxa and a higher number of silphid species compared with traps containing formaldehyde. The use of fish bait in the traps increased the number of individuals collected and the number of species collected for silphid beetles but had no effect on the collection parameters for carabids. The species composition of the carabid assemblages was minimally affected by the presence of fish bait or the type of preservative fluid, whereas the fish bait had a substantial effect on the species composition of silphids. The silphid species that feed directly on vertebrate carcasses were almost completely absent in the nonbaited traps. The results suggest that pitfall traps baited with fish and containing propylene glycol as a preservative fluid are optimal for the simultaneous sampling of carabid and silphid beetles, which both provide important ecosystem services (e.g., predation of pests and decomposition of vertebrate carcasses) and are therefore interesting for ecological research.

Thermal summation model and instar determination of all developmental stages of necrophagous beetle, Sciodrepoides watsoni (Spence) (Coleoptera: Leiodidae: Cholevinae).

Necrophagous beetles are underrepresented in forensic entomology studies despite their undeniable utility for the field. In the present article, information is presented regarding the developmental biology and instar determination of Sciodrepoides watsoni (Spence, 1813), a very common species occurring across the Holarctic region. Wild collected beetles were kept in climate chambers at constant temperature (12, 15, 18, 21 and 28 °C) and their development was regularly documented. Parameters of thermal summation models and standard errors were calculated for each developmental stage. These models may be used for an estimation of post-mortem interval in legal investigations after further validation on local populations of S. watsoni. An additional methodology is introduced for future studies of size-based characteristics, addressing instar identification bias. The methodology provided estimations (mean, standard error and standard deviation) of S. watsoni larval head capsule width for preliminary larval instar determination. The methodology may be used with other morphological features to improve instar determination accuracy.