Morphology of puparia of Megaselia scalaris (Diptera: Phoridae), a fly species of medical and forensic importance

Megaselia scalaris (Loew), a scuttle fly, is a fly species of medical and forensic importance. For use in forensic investigation, fly specimens found to associate with human corpses have to be identified at species level. Herein, we present the morphology of the puparia of the above fly species using scanning electron microscopy. The characteristic of the intersegmental spines along the dorsal and lateral segments and sculpture of the pupal respiratory horn of this puparia may be useful in future studies, to distinguish it from other closely related species.     

组织透明化技术的研究与应用

文题释义：组织透明技术：应用水溶性有机溶剂或者亲水性试剂,对固定组织通过浸泡、电泳或灌注等方式进行透化处理,然后应用高折射率介质匹配组织折射率,降低光散射,使组织达到光学透明,进而增加成像深度和图像对比度的技术。 光散射：光从一种介质进入另外一种介质时,由于介质成分的变化,光的强度和曲度都会相应改变,当遇到两种均匀介质的分界面时,光会发生反射和折射现象,但当光在不均匀介质中传播时,一部分光线会偏离原始传播路径向四面八方传播,即形成光的散射现象。 背景：对生物体进行全身细胞分析是生物医学领域的主要挑战之一。组织透明技术结合光学成像和图像处理技术,能够将整个器官或全身快速透明并进行结构和细胞分析,为在生命科学中应用先进的光学技术提供了一个非常有前景的解决方案。 目的：分析组织透明技术原理及过程,总结组织透明技术研究进展,介绍组织透明成像技术,讨论组织透明技术在生物医学研究领域中的应用。 方法：第一作者以“tissue clearing technique,tissue optical clearing,whole-body imaging,3D imaging”为关键词,检索PubMed数据库发表的相关文献,初检文献168篇,系统整理、筛选后,对纳入的72篇文献进行分析、总结和讨论。 结果与结论：目前组织透明技术主要分为两类,有机溶剂透明技术和亲水性试剂透明技术,组织透明步骤包括：①组织固定;②透化作用;③脱色作用;④折光率匹配。组织透明技术能够使组织快速光学透明,显著提高成像深度和图像对比度,结合显微成像技术如共聚焦显微镜、双光子显微镜和光片显微镜等,能够在单细胞水平实现全身或全器官的高分辨率三维成像,加速了生物体全身细胞分析进程。未来,组织透明技术将会持续发展,并将推动新型透化试剂和透化专用显微镜的研发,从而加强从完整机体系统获取结构和分子信息,有助于对完整生物系统的综合理解。 ORCID: 0000-0003-1647-7922(田婷) 中国组织工程研究杂志出版内容重点：人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程     

Surface ultrastrucure of larva and puparia of blow fly Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae)

Flies of the genus Hypopygiopsis are forensically important, as their larvae are found to associate with human corpses. In this study, the ultrastructure of larvae and puparia of Hypopygiopsis tumrasvini Kurahashi is presented using scanning electron microscopy (SEM). The larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures, i.e., antenna, maxillary palpus, and ventral organ. Two tufts of fresh outgrowths (or cerri) were observed along the dorsal margin of the mouth opening of the first instar; whereas a strong mouth hook was apparent in the second and third instars. Keilin??s organ, the sensory structure, was noticeable on the ventral surface of the thoracic segments in all instars. In the second and third instars, six conspicuous tubercles were present along the peripheral rims of the last abdominal segment. The puparia were relatively large, measuring 7.77?C9.51?mm in length and 3.10?C3.97?mm in width. At the latero-dorsal edge of the first abdominal segment, a cluster of ??125 bubble membranes was seen in young puparia; whereas a minute pupal respiratory horn was observed in old one. An SEM image revealed antler-like projections lined within a chamber of a broken pupal respiratory horn. Comparison on number of the bubble membranes of the other blow fly species was shown and the role of pupal respiratory horn compared with other dipterans was discussed.     

Tissue clearing technique: Recent progress and biomedical applications

Organisms are inherently three dimensional, thus comprehensive understanding of the complicated biological system requires analysis of organs or even whole bodies in the context of three dimensions. However, this is a tremendous task since the biological specimens are naturally opaque, a major obstacle in whole-body and whole-organ imaging. Tissue clearing technique provides a prospective solution and has become a powerful tool for three-dimensional imaging and quantification of organisms. Tissue clearing technique aims to make tissue transparent by minimizing light scattering and light absorption, thus allowing deep imaging of large volume samples. When combined with diverse molecular labeling methods and high-throughput optical sectioning microscopes, tissue clearing technique enables whole-body and whole-organ imaging at cellular or subcellular resolution, providing detailed and comprehensive information about the intact biological systems. Here, we give an overview of recent progress and biomedical applications of tissue clearing technique. We introduce the mechanisms and basic principles of tissue clearing, and summarize the current tissue clearing methods. Moreover, the available imaging techniques and software packages for data processing are also presented. Finally, we introduce the recent advances in applications of tissue clearing in biomedical fields. Tissue clearing contributes to the investigation of structure-function relationships in intact mammalian organs, and opens new avenues for cellular and molecular mapping of intact human organs. We hope this review contributes to a better understanding of tissue clearing technique and can help researchers to select the best-suited clearing protocol for their experiments.     

Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina (Diptera: Calliphoridae), and flesh fly, Liosarcophaga dux (Diptera: Sarcophagidae)

Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina, and flesh fly, Liosarcophaga dux, are presented utilizing scanning electron microscopy (SEM). Emphasis was focused on characteristic features of the puparia that could be used for differentiation from other forensically important fly species. The puparium of L. cuprina typically measures 6.2±0.2 mm in length and 2.3±0.1 mm in width. Each anterior spiracle contains a single row of 5–7 papillae. The intersegmental spines between the prothorax and mesothorax are triangular with constricted tips. A clustered bubble membrane, comprising about 30 mammillate structures, is positioned dorsolaterally on each side of the first abdominal segment in young puparia but is replaced by short, tubular respiratory horns in aged puparia. The posterior end of the puparium is broadly truncate and bears a pair of medially positioned posterior spiracles that each contains three straight spiracular slits. The puparium of L. dux is larger in comparison to L. cuprina and typically measures 9.9±0.3 mm in length and 3.8±0.2 mm in width. An anterior spiracle of this species contains 14–17 papillae. The intersegmental spines between the prothorax and mesothorax are broad and triangular. A convoluted structure of unknown function was observed at the dorsolateral edge of segments 5–11. In L. dux, the caudal segment of the puparium is slightly tapered and abruptly truncated and contains a pair of posterior spiracles that are located within a deep concavity in the terminal end. Each posterior spiracular disc appears D-shaped, with a pronounced medial projection and three vertically oriented long, narrow spiracular slits. The anatomical features presented herein may be useful for identification of fly puparia of these two species in future forensic entomological investigations.     

Differentiation between Lucilia cuprina and Hemipyrellia ligurriens (Diptera: Calliphoridae) larvae for use in forensic entomology applications

Lucilia cuprina and Hemipyrellia ligurriens are forensically important blow flies in many countries. The immature stage, in particular the third instar, of both species are generally similar in morphological appearance. In this study, the use of three main characteristics, the posterior spiracle, the dorsal spines between the first and second thoracic segments, and the cephalopharyngeal skeleton, allows for identification of both fly species, with the number of papillae on anterior spiracle being a supportive characteristic in some cases. Comparison of these characteristics is illustrated in detail.     

Morphology of immature stages of Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) for use in forensic entomology applications

In forensic investigations, all immature stages of flies (egg, larvae, and puparium) can serve as entomological evidence at death scenes. These insects are primarily used to estimate the post mortem interval (PMI), but can also be involved in the analysis of toxic substances, determining manner of death, and in indicating relocation of a corpse in homicide cases. In this study, we present the morphology of the egg, larvae, and puparium of Hemipyrellia ligurriens, a blow fly species of forensic importance in Thailand. Examination was conducted using scanning electron microscopy (SEM). The egg stage was found to display a relatively wide plastron region (or median hatch line area) that spans almost the entire length of the egg. The median hatch line is oriented in an upright position. External chorionic sculpture of the egg is present in a hexagonal pattern whose reticular boundaries are slightly elevated. In the larval stages, the most prominent morphological changes were detected upon comparison of the first to the second instar; whereas, the differences between second and third instar larvae were less obvious outside of the increase in number of posterior spiracular slits. Most of the major differences involve body size and structure of the anterior and posterior spiracles. Each anterior spiracle in both the second and third instars projects five to seven papillae apically. Each posterior spiracular disc of a third instar exhibits a complete peritreme, three spiracular slits, and a prominent button that is ventromedially located. The puparium is coarctate and features a clustered bubble membrane comprised of approximately 57 mammillate structures positioned dorsolaterally on each side of the first abdominal segment in young puparia. This feature is replaced by short, tubular respiratory horns in aged puparia. This study provides more detailed exposure of important morphological features that can be used for accurate identification of immature stages of H. ligurriens. Information presented can aid in forensic investigations involving this fly species.     

First report of human myiasis caused by Chrysomya megacephala and Chrysomya rufifacies (Diptera: Calliphoridae) in Thailand, and its implication in forensic entomology

We report a forensic entomology case associated with human myiasis in Chiang Mai Province, northern Thailand. The remains of a 53-yr-old-male were concurrently infested with third instars of the two blow fly species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), near a severe tumor lesion presented on the lower right leg. The presence of third instars, approximately 5 d old, on the day following postmortem indicated that myiasis occurred before death. This is the first report of both fly species acting as a myiasis-producing agent in Thailand. Unsynchronized data between the age of fly larvae due to myiasis premortem and verified age/ condition of the corpse suggest a potential complication and error in the estimation of postmortem interval if other predisposition fly infestations are not considered.     

Larval morphology of Chrysomya nigripes (Diptera: Calliphoridae), a fly species of forensic importance

The morphology of all instars of Chrysomya nigripes Aubertin, a blow fly species of forensic importance, is presented with the aid of both light microscopy and scanning electron microscopy (SEM). Morphological features of the cephalopharyngeal skeleton, anterior spiracle, posterior spiracle, and dorsal spines between the prothorax and mesothorax are highlighted. No consistent features were found, even using SEM, for distinguishing the first instar of C. nigripes from that of Chrysomya megacephala (F.) or Chrysomya rufifacies (Macquart), two other commonly associated blow fly species in corpses in Thailand. Several features observed in second and third instars proved to be valuable characteristics for separating these species.     

Hairy maggot of Chrysomya villeneuvi (Diptera: Calliphoridae), a fly species of forensic importance

We reported on the hairy maggot of Chrysomya villeneuvi Patton, collected from a human corpse in Thailand. Although the general morphology of the third instar closely resembled the more common hairy maggot blow fly, Chrysomya rufifacies (Macquart), the spines along the tubercles of each body segment could be used as a feature to distinguish between these species, with those of C. villeneuri bearing sharp-ended spines that encircle the entire tubercle. Not only has the resemblance of a hairy appearance between C. villeneuvi and C. rufifacies larvae been documented but also their similarity in predacious feeding behavior. The differentiation between these two species is necessary for post-mortem interval estimation in the corpse.     

Developmental rate,size, and sexual dimorphism of Megaselia scalaris (Loew) (Diptera: Phoridae): its possible implications in forensic entomology

Cosmopolitan scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae) is one of the commonest forensic species recorded colonizing human corpse indoors and in concealed environment. The occurrence of this species in such environments provides a higher evidential value to assist estimation of postmortem interval (PMI) compared to other forensically important dipterans. However, developmental and size data of M. scalaris are still lacking and they are derived from a limited range of thermal values. The objective of this study is to develop the growth model of M. scalaris by emphasizing the size range of larvae and puparia at different constant temperatures. This species was reared in six replicates at eight varying constant temperatures ranging from 23 to 36 °C and cow’s liver was provided as food source. Larvae and puparia were sampled at set time intervals and measured by their length and weight. Because interpretation of forensic entomological evidence is subject to application of different techniques, development of M. scalaris is expressed herein by using developmental table, length/morphological stage diagrams and linear/nonlinear estimation methods. From the findings, it is very important to highlight that sexual dimorphism of M. scalaris during post feeding larva and pupa stage could be observed based on size and developmental periods. Mean length and weight ratios of male to female puparia are approximately 0.8 and 0.3–0.5, respectively, indicating sexual dimorphism of this species. Developmental period in female are 4.0–11.4 h (post feeding larval stage), 3.7–24.0 h (pupal stage), and 3.0–20.1 h (total developmental period) longer in male. Due to this dimorphism, PMI estimation using M. scalaris post feeding larva or puparium specimens must be carried out carefully by to avoid inaccuracy and misinterpretation.     

Effects of temperature on development of Phormia regina (Diptera: Calliphoridae) and use of developmental data in determining time intervals in forensic entomology

Precise developmental data for forensic indicator blow fly species are essential for accuracy in the estimate of the post-mortem interval (PMI). Why, then, does the determination of the PMI result in conflicting time frames when published conspecific developmental data are used? To answer this question, we conducted constant temperature trials between the developmental minimum temperature and upper threshold temperatures (8-32 degrees C) on the forensically important blow fly species Phormia regina (Meigen) (Diptera: Calliphoridae). Flies were reared using two designs to quantify sources of variation. We measured rearing container temperatures and internal growth chamber temperatures by using thermocouples to accurately record temperatures experienced by larvae and to construct a degree-day model. Differences in experimental design, as seen across temperature studies for this fly species, did not significantly impact larval development. We also found that using set chamber temperatures rather than rearing container temperatures altered the final degree-day model. Using any minimum threshold (including an empirically determined true minimum) other than that from linear interpolation (x-intercept) violated degree-day assumptions and invalidated estimates of the PMI. We observed the minimum developmental temperature to be higher (14 degrees C) than that generated under the x-intercept method (5.46 degrees C) by using data from oviposition to adult emergence. This difference along with the noted difference in accumulated degree-days (using different base temperatures) suggests a need for additional experimentation on other forensically important fly species at low temperature thresholds to help with development of curvilinear models. Former and current estimates of the PMI may be inaccurate if the process to determine the time frame ignored degree-day model assumptions or was based upon questionable data sets.     

Forensic entomology cases in Thailand: a review of cases from 2000 to 2006

This paper presents and discusses 30 cases of cadavers that had been transferred for forensic entomology investigations to the Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University, northern Thailand, from 2000 to 2006. Variable death scenes were determined, including forested area and suburban and urban outdoor and indoor environments. The fly specimens found in the corpses obtained were the most commonly of the blow fly of family Calliphoridae, and consisted of Chrysomya megacephala (F.), Chrysomya rufifacies (Macquart) Chrysomya villeneuvi Patton, Chrysomya nigripes Aubertin, Chrysomya bezziana Villeneuve, Chrysomya chani Kurahashi, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), and two unknown species. Flies of the family Muscidae [Hydrotaea spinigera Stein, Synthesiomyia nudiseta (Wulp)], Piophilidae [Piophila casei (L.)], Phoridae [Megaselia scalaris (Loew)], Sarcophagidae [Parasarcophaga ruficornis (F.) and three unknown species], and Stratiomyiidae (Sargus sp.) were also collected from these human remains. Larvae and adults of the beetle, Dermestes maculatus DeGeer (Coleoptera: Dermestidae), were also found in some cases. Chrysomya megacephala and C. rufifacies were the most common species found in the ecologically varied death scene habitats associated with both urban and forested areas, while C. nigripes was commonly discovered in forested places. S. nudiseta was collected only from corpses found in an indoor death scene.     

Analysis of short tandem repeat (STR) polymorphisms by the powerplex 16 system and capillary electrophoresis: application to forensic practice

Allele and genotype frequencies for 15 short tandem repeat (STR) polymorphisms--D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA--in a Japanese population were estimated. No deviations of the observed allele frequency from Hardy-Weinberg equilibrium expectations were found for any of the systems studied. Between 2 new pentanucleotide STR loci, Penta E and Penta D, for which there is only limited data regarding the allelic distribution in Japanese, the Penta E locus was found to be highly polymorphic and exhibited a tri- or tetra-modal distribution pattern having allelic peaks with 5, 11, 15 and 20 repeats. The distribution was significantly different from that of the other ethnic groups. Statistical parameters of forensic importance, the power of discrimination (PD), observed and expected heterozygosity values (H), polymorphism information content (PIC), power of discrimination (PD), matching probability (pM), power of exclusion (PE), and typical paternity index (PI), were calculated for the loci. These parameters indicated the usefulness of the loci in forensic personal identification and paternity testing among Japanese. The systems Penta E, FGA, D18S51 and D8S1179 were the most informative. This method was successfully applied to forensic personal identification and paternity testing among Japanese, thereby confirming its efficacy for forensic practice.     

Observations on morphology of immature Lucilia porphyrina (Diptera: Calliphoridae), a fly species of forensic importance

Lucilia porphyrina (Walker) is a blow fly of forensic importance, and shares its geographical distribution with a related forensically important species, Lucilia cuprina (Wiedemann). The immature stages of both species are similar in general appearance; therefore, correct identification should be given special consideration. This study highlighted the main features of L. porphyrina larvae, as observed under light microscopy and scanning electron microscopy. Particular attention is given to the anterior and posterior spiracles, cephalopharyngeal skeleton, and characteristics of the dorsal spines between the prothorax and mesothorax. In the third instar specifically, morphological information on L. porphyrina showed several features that are shared by L. cuprina, and therefore need certain identification to distinguish between them. Such key features are (1) greater posterior spiracle, (2) apparent inner projection between the middle and lower slits of the posterior spiracle, and (3) strongly sclerotized peritreme. The number of papillae on the anterior spiracle may be a supplement, five to nine and three to six in L. porphyrina and L. cuprina, respectively. The key for identifying third instar of forensically important flies in Thailand has been updated to include L. porphyrina.     

Methadone blood assay by the FPIA technique: application to the monitoring of patients in maintenance treatment to opiates

A large inter-individual variability in methadone pharmacokinetics is observed in patients under maintenance treatment for major addiction to opiates. Therefore an individual dose titration of methadone is necessary, based on clinical response, i.e. symptoms of overdosage or withdrawal syndrome, but these symptoms are unspecific. However, a poor response to methadone treatment (asking for drug compliance) or the possibility of drug interactions may require the determination of methadone blood concentrations. Therapeutic drug monitoring (TDM) of those patients is performed using methadone trough blood concentrations measured by chromatography (GC or HPLC: reference methods) or by immunoassay, which gives more rapid results. A review of the literature led us to use the fluorescence polarisation immunoassay (FPIA technique) performed on a TDx-FLx analyzer. We confirmed the lack of "matrix effect" and FPIA was compared to GC-MS (gas chromatography-mass spectrometry) on patients samples. According to the literature, a methadone trough serum concentration target of 400 ng/mL is recommended; results under 100 ng/mL are considered as clinically ineffective, whereas methadone concentrations above 1000 ng/mL are frequently associated with drug toxicity. The linearity domain of the technique stays between 50 and 500 ng/mL, which is satisfactory. We describe some clinical cases from the Methadone Treatment Center of Tours (Centre Port-Bretagne), which showed that methadone blood concentration measurement may be helpful to achieve the optimal dose of methadone in each patient.     

Scanning electron microscopy of the third-instar Piophila casei (Diptera: Piophilidae), a fly species of forensic importance

The morphology of third-instar Piophila casei (L.) is described by means of scanning electron microscopy. Features of the anterior cephalic region and creeping welts that are used in larval skipping and creeping, respectively, are highlighted. Morphological features classically used for larval identification are also illustrated.     

The application of the fluorescent-antibody technique to haemagglutinating systems

The Coons' technique has been modified for the detection of antibodies on erythrocytes agglutinated by fluorescein-conjugated antisera. The results of experiments with several haemagglutinating systems suggested that specific fluorescence was found only on erythrocytes which had been agglutinated by species-specific antibodies. The significance of these findings is discussed.