Internal Morphological Analysis for Age Estimation of Blow Fly Pupae (Diptera: Calliphoridae) in Postmortem Interval Estimation*

Abstract: Larvae and pupae of blow fly species are frequently used in postmortem interval estimation, their age indicating minimum time since death. Most studies have considered age estimation of larvae, neglecting study of pupae. Relative development of external pupal features is useful, but there are also internal changes during metamorphosis that may be indicators of age, utilizing histological techniques. This study aimed to optimize preservation and histological analysis of blow fly pupae, specifically Calliphora vicina Robineau‐Desvoidy and Lucilia sericata (Meigen), and to examine internal features with potential for age estimation. Effect of hot‐water‐killing and different preservatives were examined. It was determined that blow fly pupae should be pierced through the three tagma, hot‐water‐killed, and preserved in 80% ethanol as the optimal preservation for subsequent analyses. Hematoxylin and eosin stained pupal sections revealed differences in brain and thoracic muscle development throughout the pupal stage with potential for age estimation.     

Morphology of preimaginal stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae): a comparative study

A comparative morphological study of preimaginal stages (larvae and pupae) of Calliphora vicina Robineau-Desvoidy, 1830 is presented. The entomological samples came from laboratory colonies bred under controlled environmental conditions (25°C and 60% relative humidity). In this study, a recently published technique to clear Diptera larvae for light microscopy and a standard protocol for scanning electron microscopy were used. For the morphological comparison of larval instars I, II and III, and pupae of C. vicina, different larval regions (cephalic, thoracic and abdominal, including anal division), as well the internal chitinised cephalopharyngeal skeleton, were considered separately. Our results focus on showing the changes observed throughout development for the most important structures in the cephalic region (sensilla of maxillary palpus, antennae and oral ridges), the thoracic region (the first segment and its anterior spinose band) and in the anal division of the abdominal region (posterior spiracles and shape of the papillae). In addition, some morphological structures are described or pictured for the first time, such as the ventral organ and the anterior spiracle of larva I and the antenna sensilla, Keilin's organ and wrinkled area of the anal division of all instars. The cephalopharyngeal skeleton is an important structure for the taxonomy of Diptera larvae in all instars, including Calliphoridae. Our observations in C. vicina indicate that an in-depth review of the sclerite composition is needed. Pupae and larvae stages can only be compared by following the segmentary spinose bands and the anal segment, where the morphology of the posterior spiracles and papillae can be observed, in some cases despite the reduced condition of the latter.     

Did Post-Floyd et al. Reforms Reduce Racial Disparities in NYPD Stop,Question, and Frisk Practices? An Exploratory Analysis Using External and Internal Benchmarks

Various methodological approaches to constructing external and internal benchmarks have been applied to estimate racial bias in police stop, question, and frisk (SQF) patterns. We apply an external benchmark of the race of the residential population and an internal benchmark of similarly-situated stops to estimate if racial disparities in New York City SQF data were impacted by the Floyd, et al. v. City of New York court settlement. We find that after the settlement, the racial composition of census tracts were no longer significant predictors of the stop rate after controlling for reported crime, socioeconomic factors, and police precincts. We further find that differences in SQF outcomes and hit rates between Blacks and Hispanics and similarly-situated others diminished substantially after the settlement. These findings suggest that court reforms may be an effective method for reducing racial disparities in SQF patterns.     

Identification of forensically important Chrysomya (Diptera: Calliphoridae) species using the second ribosomal internal transcribed spacer (ITS2)

The identification of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae) may be hampered by their close morphological similarities, especially as immatures. In contrast to most previous studies, the utility of a nuclear rather than mitochondrial genetic marker was investigated to solve this problem. The second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) was amplified and sequenced from all nine Chrysomya species known from Australia. Difficulties encountered with direct sequencing of ITS2 for Chrysomya flavifrons necessitated cloning prior to sequencing for this species, which revealed a low level (0-0.23%) of intraindividual variation. Five restriction enzymes (DraI, BsaXI, BciVI, AseI and HinfI) were identified that were able to differentiate most members of the genus by polymerase chain reaction (PCR) restriction fragment length polymorphism (PCR-RFLP). The PCR-RFLP analysis revealed characteristic restriction profiles for all species except the closely related species pairs Chrysomya latifrons+Chrysomya semimetallica and Chrysomya incisuralis+Chrysomya rufifacies. Ch. incisuralis and Ch. rufifacies were able to be separated using the size differences resulting from amplification of the entire ITS region. The lack of intraspecific ITS2 sequence variation among eight Ch. incisuralis specimens was verified by the identical restriction profiles generated from these specimens. A DNA-based approach, such as PCR-RFLP, has the capacity to be useful for the identification of forensic entomological evidence in cases where morphological characters are unreliable.     

大鼠弥漫性脑损伤神经元HSP70表达及神经髓鞘的病理变化

目的探讨脑神经元HSP70表达变化和神经髓鞘病变在弥漫性脑损伤(DB I)诊断及其损伤时间判断的价值。方法将50只SD大鼠分为正常对照组和DB I组,后者按DB I后处死大鼠的时间不同又分为0.5h、1h、3h、6h、12h、1d、3d、7d及10d组。然后复制大鼠DB I模型,脑组织常规取材后进行HSP70免疫组化及劳克坚牢蓝(LFB)髓鞘染色,观察神经元HSP70的变化和神经髓鞘病变。结果大鼠DB I后0.5h,脑干及海马处见HSP70免疫组化染色阳性的神经元;伤后1h明显,12h达到高峰,1d下降,7d基本恢复正常。神经髓鞘于伤后0.5h开始逐渐出现水肿,分层、碎裂及聚集成团块状,伤后1d最明显,3d后开始逐渐减退,10d基本正常。结论神经元HSP70表达变化和神经髓鞘病变程度对DB I诊断及其损伤时间判断具有一定的参考价值。     

Could NF-kappaB and caspase-3 be markers for estimation of post-interval of human traumatic brain injury?

BACKGROUND: Previous studies in a rat model showed expressions of caspase-3 (p20) and NF-kappaB (p65) in different time after TBI. We test the activation changes of caspase-3 and NF-kappaB of human specimen after traumatic brain injury (TBI) in this study to see whether they could be markers to estimate post-intervals of TBI for forensic practice. MATERIAL AND METHODS: Caspase-3 (p20) and NF-kappaB (p65) immunohistochemical staining were performed on 39 TBI specimens (dead from TBI) and eight controls (dead from none TBI reasons) grouped 0, 12, 24, 48, 72, 168, 264 and 480 h. Positive cells were counted and image analysis techniques were used to determine the morphological changes in each group. RESULTS: Immunohistochemical analysis showed a marked induction of caspase-3 (p20) in every injured group compared with normal controls; each positive group has it's morphological character with variant positive cell count. NF-kappaB (p65) did not showed markedly change compared with normal controls until 168 h after TBI and almost all nerve cells were stained by NF-kappaB (p65) in 264 and 480 h groups. CONCLUSION: NF-kappaB (p65) could contribute to estimate delayed death cases after TBI for forensic applications. Caspase-3 (p20) estimating post-TBI intervals are dubious but could suggest TBI exists.     

眶下裂和眶下沟横断面CT表现及其解剖基础研究

目的研究正常人眶下裂和眶下沟横断面CT表现及其解剖基础,避免将其误认为眶壁骨折。方法正常颅骨25个,肉眼观察眶下裂和眶下沟解剖形态。5个颅骨和20名正常人,眶横断面CT扫描,观察眶后外侧形态,15例辅以多平面重建和三维立体重建。结果在标本和正常人横断面上,眶后外侧面同时反映眶外侧壁和大部分眶下壁,其表现有:(1)单处缺裂,两侧对称或不对称;(2)双处缺裂,缺裂之间夹有片状小骨,小骨长轴与眶外侧壁平行一致;(3)类似双处缺裂,缺裂之间出现片状小骨,小骨呈锥状,长轴在前后方向上。大体标本和眼眶重建图像显示,上述三种情况分别对应的解剖基础是:(1)棒球杆样眶下裂;(2)V形眶下裂,即眶下裂分内外两支,内支位于蝶骨大翼下缘与下颌骨后缘间,外支位于蝶骨大翼下缘与颧骨之间,内外支间形成一向上开口的“V”字形;(3)深陷的眶下沟,伴有突出并向内倾斜的外侧壁。结论熟悉正常人眶下裂和眶下沟的横断面CT表现及其解剖学基础,可以避免将其误认为眶壁骨折。     

The Use of CT Scans in Estimating Age at Death by Examining the Extent of Ectocranial Suture Closure

We examined the utility of CT scans in the evaluation of degree of ectocranial suture closure. Five cranial points (left and right midcoronal positions, left and right midlambdoidal positions, and the lambda) were evaluated in 231 CT scans using a three‐point scoring system (open, partial closed, and closed). The slice increment and thickness varied between three groups of the scans. The results showed a good correlation between degree of suture closure and increasing age in each group. Young individuals (<40 years) and old individuals (>60 years) could be clearly distinguished from the middle‐aged individuals. ANOVA test revealed no difference between two groups of scans and between left and right sides (p > 0.05). Interobserver agreement was good, especially considering the score by sides. The results of this study create the base for developing a robust and simple method to estimate the age at death using CT scans.     

Small business innovation: firm level evidence from Sweden

This paper examines innovation among very small firms and provides new insights into both internal and external determinants of patenting. Applying a non-linear panel data approach to about 160,000 observations on manufacturing firms in Sweden for the period 2000?C2006, the following facts emerge: (i) in contrast to larger firms, innovation in micro firms with 1?C10 employees is not sensitive to variation in internal financial resources, (ii) skilled labour is even more important for innovation among micro firms compared to other firms, (iii) affiliation to a domestically owned multinational enterprise group increases the innovation capacity of small businesses, (iv) small firms?? innovation is closely linked to participation in international trade and exports to the G7-countries, and (v) there is no statistically significant evidence that proximity to metropolitan areas, or presence in a specialized cluster, increases the innovativeness of the smallest firm.     

Alcohol-Impaired Driving in Norway and Sweden: Another Look at "The Scandinavian Myth"

Ross's (1975) assessment of the Scandinavian drinking-and-driving laws was a pivotal paper in the international literature because, in many ways, Norway and Sweden offer a critical test of the "tough approach" to deterring alcohol- impaired drivers. The present review raises some methodological and philosophical questions about Ross's approach to the Scandinavian data and examines four types of indirect evidence for deterrence in Norway and Sweden. While no conclusive evidence for deterrence could be identified, there was internal consistency in the pattern of circumstantial evidence pointing to a workable system of external restraints and internal controls in Scandinavia; and this pattern of evidence contrasted sharply with that obtained in the U.S.     

牙齿结构变化图像分析测量指标与个体年龄相关性的研究

用图像分析法对牙齿结构变化指标与个体年龄相关性进行研究,所用牙齿标本均取自香港大学口腔生物中心牙库,共得到人体牙齿842颗,标本来源的个体年龄范围为13～84岁,采用Kontron电子计算机图像分析仪直接测量法进行各牙齿标本的外表和切片检查,检查所得数据均输入电子计算机数据库软件,并使用社会科学统计专用软件包(spss)进行相关性分析。结果表明,与个体年龄相关性较好的指标依次为TP2’,S2,T2’,EP,TCI等,如按牙齿类型分类,与个体年龄相关性较好的指标为:T2’,S2’,TCI等(切牙与尖牙类),CCP’,EP’,E’等(前磨牙类),TP2’,S2’,EPWNL’等(磨牙类)。     

A comprehensive regression tree to estimate body weight from the skeleton

The purpose of this research is to estimate actual body weight (in particular obesity) from the human skeleton. Known individuals (total n=187) were studied from the Bass Collection from the University of Tennessee, Knoxville. This research combines bone density, cross-sectional geometry of the femur and skeletal pathologies. Bone mineral density was calculated for the proximal femur. Three-dimensional bone surface models were created from computed tomographic scans. Cross-sectional geometry of the femur was calculated at five locations along the diaphysis. The pathologies analyzed were heel spurs, diffuse idiopathic skeletal hyperostosis (DISH), and tibial osteoarthritis. The best regression tree model included only four variables. The first split to estimate body weight was the minimum moment of inertia (I(y) ) at the distal femur, the second was midshaft width, then anteroposterior thickness at the proximal slice and the final split was the degree of DISH (SD 17.1-31.0 kg). The ability to estimate body weight from the skeleton is one more useful tool for the osteologist.     

Forensic toxicological study on adipocere formation in submerged cadavers of female albino rats intoxicated with cadmium

There is a dearth of information on the mutual interaction between metal intoxication and adipocere formation. Herein, 40 adult female albino rats were distributed into two equal groups, one used as control while the other orally administered single dose of cadmium chloride (CdCl₂) 225 mg/kg·bw (LD_min). Control group was killed by cervical dislocation. Half of dead rats of both groups were subjected for determination of iodine value and estimation of cadmium (Cd) residues while the other half of both groups were submerged in opened glass container previously filled with 4 L dechlorinated tap water kept in closed room with an open air access (one cadaver/container). Gross morphological changes of submerged cadavers were recorded weekly along the experiment. At the end of the experiment, after 3 months, samples were collected again for iodine value determination and estimation of Cd residues. The obtained results revealed the depressant effect of Cd toxicity on development of adipocere. Cd residues were found in different tissues of cadavers at time of death with the highest amount in the intestines followed by the liver and kidneys, then lungs, adipose tissue, muscles, and finally the bones. After 3 months of water submersion, tissues exhibited significant decrease in the amount of Cd residues but to a limit that was still detected. This study concluded the possibility of detection of Cd residues even after adipocere formation. Additionally, it shed light on the possibility of the interference of environmental pollution with the natural rate of decomposition especially adipocere formation.     

Changes in the regulation of iodine crystals and chemical mixtures containing over 2.2 percent iodine. Final rule

This rulemaking changes the regulation of the listed chemical iodine under the chemical regulatory provisions of the Controlled Substances Act (CSA). The Drug Enforcement Administration (DEA) believes that this action is necessary to remove deficiencies in the existing regulatory controls, which have been exploited by drug traffickers who divert iodine (in the form of iodine crystals and iodine tincture) for the illicit production of methamphetamine in clandestine drug laboratories. This rulemaking moves iodine from List II to List I; reduces the iodine threshold from 0.4 kilograms to zero kilograms; adds import and export regulatory controls; and controls chemical mixtures containing greater than 2.2 percent iodine. This rulemaking establishes regulatory controls that will apply to iodine crystals and iodine chemical mixtures that contain greater than 2.2 percent iodine. This regulation therefore controls iodine crystals and strong iodine tinctures/solutions (e.g., 7 percent iodine) that do not have common household uses and instead have limited application in livestock, horses, and for disinfection of equipment. Household products such as 2 percent iodine tincture/solution and household disinfectants containing iodine complexes will not be adversely impacted by this regulation. Additionally, the final rule exempts transactions of up to one-fluid-ounce (30 ml) of Lugol's Solution. Persons handling regulated iodine materials are required to register with DEA, are subject to the import/export notification requirements of the CSA, and are required to maintain records of all regulated transactions involving iodine regardless of size.     

The Lateral Angle Revisited: A Validation Study of the Reliability of the Lateral Angle Method for Sex Determination Using Computed Tomography (CT),

This article presents the results of a validation study of a previously published method of sex determination from the temporal bone. The purpose of this study was to evaluate the lateral angle method for the internal acoustic canal for accurately determining the sex of human skeletal remains using measurements taken from computed tomography (CT) scans. Previous reports have observed that the lateral angle size in females is significantly larger than in males. The method was applied to an independent series of 77 postmortem CT scans (42 males, 35 females) to validate its accuracy and reliability. The mean lateral angle of the internal acoustic canal was found to be larger in females (46.5°) than in males (43.4°). However, the difference was not statistically significant and the sex differences reported in previous studies were not substantiated. In light of the observed results, the lateral angle method appears to be of minimal practical use in forensic anthropology and archeology.     

根据胸骨形态变化推算年龄的研究

<正> 本文运用多元线性回归分析的方法,对74例成人汉族男性胸骨(19～48岁)的形态变化与年龄的关系进行了研究。通过胸骨上选定的7个形态变化标志点,依其各自的形态变化规律,制定出相应的等级评分标准。根据此标准对74例胸骨标本进行了观察评分。然后将所得原始数据和年龄一起输入电子计算机,求出由胸骨形态变化,计算年龄的多元回归方程: =10.78+0.82X_1+0.85X_2+0.80X_3+0.38X_4+2.51X_5+0.64X_6+3.02X_7±2.45 经方差分析表明P<0.01,回归高度显著。本文提出的胸骨判定年龄的方法,在无名尸骨的法医学鉴定中,具有实用价值。     

Forensic Facial Approximation: Study of the Nose in Brazilian Subjects

Forensic facial approximation is an auxiliary method for human identification and allows facial recognition. The midface, that includes the nose, is vital for the recognition of a familiar face. The purpose of this study was to set hard tissue parameters to estimate nasal width, to test the method to estimate nasal width of Brazilians, and to analyze the relationship between nasal profile and facial type. A total of 246 computed tomography scans (183 females and 63 males) of adults were analyzed in Horos. Bone tissue measurements and facial type classification were performed on the skull scan. Nasal profile morphology was accessed through the tool 3D surface rendering. There was a difference around 3 mm from real to predicted nose through the method to estimate nasal width in Brazilians. So, the method may be used in forensic practice. Straight nose was associated with long face type.     

Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram.

Developmental behavior of eggs, larva and pupa of the blowfly species Lucilia sericata (Meigen) were studied under 10 different temperature regimes. Data from these studies were used to construct the isomegalen-diagram. In this diagram, time from hatching to peakfeeding is plotted against temperature, each line representing identical larval length at various temperatures. If the temperature is roughly constant, as is the case with corpses found indoors, the age of the maggot can be read off instantly from its length, provided that the maggot has not entered the migratory phase. Where temperature is variable, an age range can be estimated between the points where the measured larval length cuts the graph at the maximum and minimum temperatures recorded. Equally, the isomorphen-diagram representing all morphological stages from oviposition to eclosion should be used, if maggots in the migratory phase or pupae or puparia are recovered from the scene. The isomegalen- and the isomorphen-diagrams could facilitate a quick and more precise estimate of the postmortem interval even for the inexperienced investigator. In addition, our results vary from those of other investigators, suggesting a different thermal behavior of the holarctic blowfly L. sericata in various zoogeographic regions.     

Study of steroidogenesis in pupae of the forensically important blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae)

Protophormia terraenovae is a forensically important fly whose development time is studied by forensic entomologists to establish the time elapsed since death (post-mortem interval, PMI). Quantity and nature of ecdysteroid hormones present in P. terraenovae pupae were analysed in order to determine if they could be correlated to the age of pupae found on corpses and thereby could give information on the PMI. Ecdysteroid levels were quantified during the pupal-adult development of synchronised animals using enzyme immunoassay (EIA), a sensitive method allowing acurate quantification in one pupa. Two types of pupae were compared: "fresh" pupae, kept frozen until analysis and "experimentally dried" pupae, which were left for several weeks at ambient temperature. A peak of ecdysteroids was detected between 36 and 96 h after pupariation in fresh animals. It was not observed in "experimentally dried" pupae. High-pressure liquid chromatography (HPLC) analyses combined with EIA showed that 20-hydroxyecdysone (20E) was the major free ecdysteroid at various pupal ages. Enzymatic hydrolysis experiments revealed the presence of apolar conjugates at all ages tested. However, neither qualitative nor quantitative difference was detected between early and late pupae. This study gives precise information on the nature and quantity of ecdysteroids in the course of pupal development of a calliphorid fly. The limits of using ecdysteroid measurement as a tool in forensic entomology are discussed.