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.

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.

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.

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.

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.

Revision of Palearctic and Oriental Necrophila Kirby et Spence, part 3: subgenus Calosilpha Portevin (Coleoptera: Silphidae: Silphinae).

A taxonomic revision of the subgenus Calosilpha Portevin, 1920 (of the genus Necrophila Kirby & Spence, 1828) through Asia is presented. Four valid species are recognized: (1) Necrophila (C.) brunnicollis (Kraatz, 1877), widely distributed from Bhutan and China: Yunnan province to Far East of Russia and Japan (including Ryukyu Islands); (2) N. (C.) cyaneocephala (Portevin, 1914), endemic to Taiwan; (3) N. (C.) cyaniventris (Motschulsky, 1870), widely distributed from northern India to Vietnam and southern China (east to Hainan Island); and (4) N. (C.) ioptera (Kollar & Redtenbacher, 1844), comb. nov. (ex Calosilpha), widely distributed in Pakistan, India (Himachal Pradesh to Assam) and Nepal. Eusilpha (Calosilpha) bicolor imasakai Nishikawa, 1986 and Eusilpha (Calosilpha) kurosawai Nishikawa, 1986 are confirmed as junior subjective synonyms (sensu Cho & Lee 1995, Ji 2012) of Silpha brunnicollis Kraatz, 1877. Description of important adult taxonomic characters (including male genitalia) and a key to species is included. Georeferenced records for all four species are mapped. First reliable records of Necrophila (Calosilpha) brunnicollis from China: Hong Kong and Anhui, Fujian, Gansu, Hubei, Jiangxi, Jilin, Qinghai and Zheijiang provinces and Guangxi autonomous region; N. (C.) cyaniventris from India: Himachal Pradesh, Uttarakhand, Meghalaya, Arunachal Pradesh, Nagaland and Manipur; and N. (C.) ioptera from Pakistan, India: Uttarakhand, Uttar Pradesh, Bihar, Sikkim, West Bengal and Assam are given (only imprecise records from "Himalaya" had been published for the last two species). First records of N. (C.) brunnicollis from Bhutan, and of N. (C.) cyaniventris from China: Hainan and Yunnan provinces, Myanmar, Laos and Cambodia are given. Parsimony analysis supports the monophyly of Calosilpha, with two clades, one formed by N. (C.) brunnicollis and N. (C.) cyaneocephala and the other by N. (C.) cyaniventris and N. (C.) ioptera. Geometric morphometrics discriminated four taxa of Necrophila (Calosilpha). Results indicated a sexual dimorphism between sexes in all species. Shape variability was concluded between the taxa. Populations of N. (C.) brunnicollis from continental Asia and three regions of Japan (Honshu + Shikoku, Kyushu and Ryukyus) also were examined. MANOVA was significant and supported shape differences in male and female populations. When testing each pair of groups by discriminant analysis, only differences between male populations from Japan (Kyushu) and those from Japan (Honshu + Shikoku) showed insignificance.

Taxonomic revision of genus Ablattaria Reitter (Coleoptera, Silphidae) using geometric morphometrics.

The genus Ablattaria Reitter, 1884 (Coleoptera: Silphidae: Silphinae) is revised. Four taxa are recognized as valid species: Ablattariaarenaria (Kraatz, 1876), Ablattariacribrata (Ménétries, 1832), Ablattarialaevigata (Fabricius, 1775) and Ablattariasubtriangula Reitter, 1905. Ablattarialaevigatavar.meridionalis Ganglbauer, 1899 is newly treated as a junior subjective synonym of Ablattarialaevigata. Lectotypes are designated for Phosphugaarenaria Kraatz, 1876, Ablattariaarenariavar.punctigera Reitter, 1884, Ablattariaarenariavar.alleoni Portevin, 1926, Silphacribrata Ménétries, 1832, Silphalaevigata Fabricius, 1775, Silphagibba Brullé, 1832, Ablattariagibbavar.costulata Portevin, 1926, Ablattariagibbavar.distinguenda Portevin, 1926, Ablattariagibbavar.punctata Portevin, 1926 and Ablattariasubtriangula Reitter, 1905. The distribution of all taxa is mapped, based on material examined. Geometric morphometric methods were used to evaluate shape variability in Ablattaria. Results indicated sexual dimorphism in all species. Shape inconsistency was found between the sexes of all taxa when tested independently. The first two relative warp axes indicated 65.17% shape variation in males and 65.72% in females. Canonical variate analysis separated the taxa studied. There was minimal overlap between some groups in both sexes. Differences in body shape between populations of Ablattarialaevigata from Central Europe, Italy and Greece + Turkey were also examined. Relative warps implied 58.01% shape variability on both axes in males and 64.78% in females. CVA revealed noticeable overlaps between the groups, although the Italian population demonstrated a higher separation in both sexes.