Identification of forensically important fly eggs using a potassium permanganate staining technique

Fly eggs found in corpses can be utilized as entomological evidence in forensic investigations of deaths if the species of fly and the developmental rate at a temperature similar to the death scene are known. The species identification of fly eggs is particularly important, and previously, scanning electron microscope has been used for this purpose. Herein, we report a simple technique, using light microscopy, to identify forensically important eggs of Chrysomya rufifacies (Macquart), Chrysomya megacephala (Fabricius), Chrysomya pacifica Kurahashi, Chrysomya nigripes Aubertin, Aldrichina grahami (Aldrich), Lucilia cuprina (Wiedemann), Musca domestica L. and Megaselia scalaris (Loew). A 1% potassium permanganate solution was used to stain egg surfaces for 1 min, followed by dehydration in 15, 70, and 95%, absolute alcohol (each solution for 1 min) and the eggs were permanently mounted. The characteristics are based on the width of plastron, morphology of plastron area surrounding the micropyle and chorionic sculpturing, with the length of egg being used as supplemental feature.     

Ultramorphological characteristics of Chrysomya megacephala (Diptera, Calliphoridae) eggs and its eclosion

Chrysomya blowflies are originally from Africa and Australasia and were introduced in the American Continent, probably as a sheep parasite, in the 1970s. These flies are extremely important for medical-sanitary purposes and are useful to forensic entomology being used as an indicative of decomposition time in human corpses. The morphology of the larval and pupal stages of some species has been already studied by different authors demonstrating that it is possible to identify them in premature stages. In this study, Chrysomya megacephala egg ultramorphology was analyzed to describe its structure, generating data for further comparison between different species and genera. The blowflies were collected in Rio Claro city, SP, Brazil. Flies were attracted by fish carcasses and maintained in net cages; small portions of minced meat were placed in plastic pots for egg laying. Eggs were collected, fixed in alcoholic Bouin, prepared according to SEM routine and observed under Philips scanning electron microscope. C. megacephala eggs are oval with one flat face and another convex, measuring approximately 0.52 (+/-0.03) mm of length and 0.12 (+/-0.02) mm of width. The micropyle is circular in shape, with two perforations enclosed by countless small projections and is located in one of the extremities in the interface between the flat and convex faces. On the convex surface hexagonal imprints were observed, this surface is impermeable to water and gases. The flat surface has numerous round projections with different sizes, creating a permeable surface with a thinner chorion from which the larvae hatches. The larvae presents itself all wrinkled and with numerous cuticular projections with a thorn shape facing the posterior region. The cuticle projections with a thorn shape from C. megacephala larvae are probably a vestige of the thorns found in larvae of parasite Diptera.     

Sound radiation around a flying fly

Many insects produce sounds during flight. These acoustic emissions result from the oscillation of the wings in air. To date, most studies have measured the frequency characteristics of flight sounds, leaving other acoustic characteristics--and their possible biological functions--unexplored. Here, using close-range acoustic recording, we describe both the directional radiation pattern and the detailed frequency composition of the sound produced by a tethered flying (Lucilia sericata). The flapping wings produce a sound wave consisting of a series of harmonics, the first harmonic occurring around 190 Hz. In the horizontal plane of the fly, the first harmonic shows a dipolelike amplitude distribution whereas the second harmonic shows a monopolelike radiation pattern. The first frequency component is dominant in front of the fly while the second harmonic is dominant at the sides. Sound with a broad frequency content, typical of that produced by wind, is also recorded at the back of the fly. This sound qualifies as pseudo-sound and results from the vortices generated during wing kinematics. Frequency and amplitude features may be used by flies in different behavioral contexts such as sexual communication, competitive communication, or navigation within the environment.     

Developmental changes of poly(ADP-ribose) polymerase expression in Sarcophaga peregrina

To elucidate the physiological role of poly(ADP-ribose) polymerase (PARP), we studied the levels of PARP mRNA and protein during the developmental stages of Sarcophaga peregrina. PARP mRNA expression changed remarkably throughout the developmental stages. The level of PARP mRNA (the molecular ratio of PARP mRNA to the total RNA) was highest in unfertilized eggs and that of PARP protein (the molecular ratio of PARP protein to the total protein of the crude extract) was high in unfertilized and fertilized eggs and in 1st instar larvae. During the embryogenesis period, the levels of PARP mRNA and protein gradually decreased. The levels of PARP mRNA during larval and pupal periods became less than about 5% of that in unfertilized eggs. After the emergence of adult flies, the levels of PARP mRNA and protein increased both in female and male flies. PARP activity normalized with the total amount of protein in the crude extract changed in parallel to the level of PARP protein throughout the developmental stages. The biological significance of the drastic change of mRNA and protein levels of PARP still remains to be clarified.     

Are the evidences of forensic entomology preserved in ethanol suitable for SEM studies?

In forensic practice, the use of arthropod evidences to estimate the postmortem interval is a very good approach when the elapsed time from death is long, but it requires the correct identification of the specimens. This is a crucial step, not always easy to achieve, in particular when dealing with immature specimens. In this case, scanning electronic microscopy (SEM) can be useful, but the techniques used to preserve specimens in forensic practice are usually different from those used to prepare specimens for SEM studies. To determine whether forensic evidences preserving techniques are also compatible with SEM analysis, we have compared specimens of all the immature stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae) preserved in 70% ethanol, with others prepared with aldehydic fixative techniques that are more appropriate for SEM studies. At the same time, two drying techniques have also been compared with both fixative techniques, the critical point drying and air-drying following with hexamethyldisilizane treatment (HMDS). Our results indicate that there are not basis against recommending the use of ethanol to preserve forensic entomological evidences and that both drying methods appear to offer good results for second and third instar larvae, although HMDS behaves better with eggs and pupae.     

Increased dietary cholesterol enhances cold tolerance in Drosophila melanogaster

For many years, non-freezing cold shock injury has been associated with damage to the cell membrane. In this study, we enhanced membrane cholesterol levels of Drosophila melanogaster by raising larvae on a cholesterol-augmented diet. Diet augmentation significantly increased the amount of cholesterol in the cell membranes of the adult flies (1.57+/-0.17 nmol per mg vs. 0.93+/-0.11 nmol per mg). Flies on the cholesterol-augmented diet exhibited a greater intrinsic cold tolerance: this group had a higher survival rate after a 2-h cold shock of -5 degree C than did the control group (71.0+/-6.6 percent vs 36.0+/-8.1 percent). Cholesterol-augmented flies also had a significantly greater capacity to rapidly cold-harden to -7 degree C (36.7+/-4.4 percent) compared to flies on a control diet (20.0+/-2.9 percent). These results suggest a mechanistic link between protection from chilling or cold shock injury and modifications to the cellular membrane.     

An electric field screen prevents captured insects from escaping by depriving bioelectricity generated through insect movements

An insulated conductor wire (ICW) paralleled with an earthed net was used to observe movements by vinegar flies in relation to their electricity release. ICW was negatively charged to create a positive charge on the net. At particular voltages, flies were attracted to ICW. This attraction was triggered by the deprivation of the insect negative charge with the net. Eventually the insects became net positive and were drawn to the ICW negative charge. The attracted insects generated bioelectricity through skeletal muscular movements. However, the electricity produced was depleted by the net without neutralizing their positive charge in the insect body.     

Sub-zero cooling synchronizes post-diapause development of codling moth,Cydia pomonella

Sub-zero cooling treatments of -10 degree C and -15 degree C for 2-6 days were evaluated as a means of meeting the chilling requirement of diapause and to synchronize post-diapause development in larvae which were held in diapause for less than 6 months. Sub-zero cooling did not affect male longevity. After six months in diapause, diapause/cooled females were longer lived than diapause control females but not longer than non-diapause females. In general, diapause-cooled males passed more spermatophores than males from control diapause and non-diapause groups. In general, sub-zero cooling did not consistently affect fecundity and % egg hatch. Non-diapause females laid the most eggs after six months in diapause, and diapause-cooled females laid the most eggs after seven months in diapause. The duration of sub-zero cooling had a significant effect on post-diapause emergence in relation to the duration that the larvae were in diapause. Sub-zero cooling for 4 days at -10 degree C significantly reduced the number of days to adult emergence of larvae which had been in diapause for 0-4 months. Sub-zero cooling at -15 degree C for durations of 2, 4, and 6 days had more variable effects on emergence, but in most cases, sub-zero cooling reduced the amount of time to and span of adult emergence.     

The early stages of three Palaearctic species of saproxylic hoverflies (Syrphidae, Diptera)

The third stage larva and puparium of the cerioidine hoverflies, Ceriana vespiformis (Latreille, 1804) and Sphiximorpha subsessilis (Illiger in Rossi, 1807) and the milesine, Spilomyia digitata (Rondani, 1865) (Diptera, Syrphidae) are described from specimens collected in the field. Ceriana vespiformis and S. digitata larvae were obtained from wet, decaying roots and tree holes of Fraxinus angustifolius L., respectively, in Spain and S. subsessilis from an exudation of sap on an Abies alba Miller tree in France.     

Circulating hemocytes from larvae of Melipona scutellaris (Hymenoptera,Apidae, Meliponini): Cell types and their role in phagocytosis

Infection in insects stimulates a complex defensive response. Recognition of pathogens may be accomplished by plasma or hemocyte proteins that bind specifically to bacterial or fungal polysaccharides. Several morphologically distinct hemocyte cell types cooperate in the immune response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping them in hemocyte aggregates called nodules, or by forming an organized multicellular capsule around large parasites. In the current investigation the cellular in the hemolymph third instar larvae of M. scutellaris has been characterized by means of light microscopy analysis and phagocytosis assays were performed in vivo by injection of 0.5 μm fluorescence beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found in 3rd instar larvae: prohemocytes, plasmatocytes, granulocytes and oenocytoids. The results presented plasmatocytes and granulocytes involved in phagocytic response of foreign particles in 3rd instar larvae of M. scutellaris.     

Ultrastructural analysis of midgut cells from Culex quinquefasciatus (Diptera: Culicidae) larvae resistant to Bacillus sphaericus

The larvicidal action of the entomopathogen Bacillus sphaericus towards Culex quinquefasciatus is due to the binary (Bin) toxin present in crystals, which are produced during bacterial sporulation. The Bin toxin needs to recognize and bind specifically to a single class of receptors, named Cqm1, which are 60-kDa α-glucosidases attached to the apical membrane of midgut cells by a glycosylphosphatidylinositol anchor. C. quinquefasciatus resistance to B. sphaericus has been often associated with the absence of the α-glucosidase Cqm1 in larvae midgut microvilli. In this work, we aimed to investigate, at the ultrastructural level, the midgut cells from C. quinquefasciatus larvae whose resistance relies on the lack of the Cqm1 receptor. The morphological analysis showed that midgut columnar cells from the resistant larvae are characterized by a pronounced production of lipid inclusions, throughout the 4th instar. At the end of this stage, resistant larvae had an increased size and number of these inclusions in the midgut cells, while only a small number were observed in the cells from susceptible larvae. The morphological differences in the midgut cells of resistant larvae found in this work suggested that the lack of the Cqm1 receptor, which also has a physiological role as being an α-glucosidase, can be related to changes in the cell metabolism. The ultrastructural effects of Bin toxin on midgut epithelial cells from susceptible and resistant larvae were also investigated. The cytopathological alterations observed in susceptible larvae treated with a lethal concentration of toxin included breakdown of the endoplasmic reticulum, mitochondrial swelling, microvillar disruption and vacuolization. Some effects were observed in cells from resistant larvae, although those alterations did not lead to larval death, indicating that the receptor Cqm1 is essential to mediate the larvicidal action of the toxin.This is the first ultrastructural study to show differences in the cell morphology of resistant larvae and further investigation is needed to understand the impact of the lack of expression of midgut enzymes on the physiology of resistant insects.     

Insect wings as retrospective/accidental/forensic dosimeters: An optically stimulated luminescence investigation

Estimation of the radiation released during nuclear accidents or radiological terrorist events is imperative for the prediction of health effects following such an exposure. In addition, in several cases there is a need to identify the prior presence of radioactive materials at buildings or sites (nuclear forensics). To this direction, several materials have been the research object of numerous studies the last decade in an attempt to identify potentially new retrospective/accidental/forensic dosimeters.However, the studies targeting biological materials are limited and their majority is mainly focused on the luminescence behavior of human biological material. Consequently, the use of such materials in retrospective dosimetry presumes the exposure of humans in the radiation field. The present work constitutes the first attempt to seek non-human biological materials, which can be found in nature in abundance or in/on other living organisms. To this end, the present work investigates the basic optically stimulated luminescence behavior of insect wings, which exhibit several advantages compared to other materials. Insects are ubiquitous, have a short life expectancy and exhibit a low decomposition rate after their death.Findings of the present study are encouraging towards the potential use of insects' wings at retrospective/accidental/forensic dosimetry, since they exhibit linear OSL response over a wide dose range and imperceptible loss of signal several days after their irradiation when they are kept in dark. On the other hand, the calculated lower detection limit is not low enough to allow their use as emergency dosimeters when individuals are exposed to non-lethal doses. In addition, wings exhibit strong optical fading when they are exposed to daylight and thus special care should be taken during the sampling procedure in order to use the wings as accidental/forensic dosimeters, by seeking (dead) insects in dark places, such as behind furniture, equipment or in air-shafts.Finally, a new single aliquot measurement protocol is also successfully tested on the wings for the dose estimation, while further work is in progress to validate it on other (heat-sensitive) materials as well.     

On the Metabolism of Dipterex

The metabolism of Dipterex in mammals, insects, plants, and microorganisms is discussed. Based on studies with P³²- and C¹⁴- labelled Dipterex the degradation of the insecticide and the different metabolic pathways are indicated. Whereas the detoxification of Dipterex in mammals and in cotton plant proceeds mainly via hydrolysis of the phosphonate C–P bond, its degradation in microorganisms invokes only O–CH₃ ester cleavage. In Prodenia larvae, Dipterex is metabolized preferentially (70%) by hydrolysis of O–methyl ester linkages and to a minor extent (30%) by splitting of C–P bond.     

Circulating hemocytes from larvae and adults of Carabus (Chaetocarabus) lefebvrei Dejean 1826 (Coleoptera, Carabidae): cell types and their role in phagocytosis after in vivo artificial non-self-challenge

Carabus lefebvrei Dejean 1826 is an helicophagous Italian endemic ground beetle that lives in central and south Apennines mountain forests, from lower altitudes to about 1500 m. In ground beetles, no morphofunctional data about immune system is available, even though they are well known both taxonomically and ecologically and they have been often used as indicators of the habitat quality due to their specificity to certain habitat types. In the current investigation the cellular population in the hemolymph of adult and third instar larvae of C. lefebvrei has been characterized by means of light and electron microscopy analysis and phagocytosis assays were performed in vivo by injection of 0.9 microm carboxylate-modified polystyrene latex beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found both in larvae and in adults: prohemocytes, granulocytes, oenocytoids and plasmatocytes. After in vivo artificial non-self-challenge treatments, C. lefebvrei showed a non-specific immune response involving phagocytosis performed by plasmatocytes, both in adults and in larvae and by oenocitoids in larvae. In untreated animals, the hemocyte type presenting a firm phagocytic activity, the plasmatocytes, presented a percentage significantly higher in larvae than in adults, and after latex beads injections in larvae there was a tendency of significant difference in plasmatocyte percentage compared to controls injected with phosphate saline buffer. We think that these differences could be correlated with the peculiar morphology (less chitinization) and ecology of larval stages that are more sensitive to pathogens than adults.     

The effect of cryopreservation process on morphology and fertilising ability of Japanese quail (Coturnix japonica) spermatozoa

Changes in the morphology and fertilising ability of Japanese quail spermatozoa were studied after semen dilution, equilibration and freezing-thawing process in order to determine the optimal diluent, cryoprotectant and the freezing-thawing method. Subsequent stages of quail semen cryopreservation caused significant decline in spermatozoa morphology and their ability to fertilise the ovum. Semen dilution with Lake's extender alone reduced the number of morphologically normal spermatozoa and decreased their fertilising ability. Dimethylacetamide (DMA) was the least detrimental but equilibration of quail spermatozoa with this cryoprotectant caused further decline in the number of morphologically normal cells. However, despite these changes, after artificial insemination with semen equilibrated with DMA 25.8 percent of fertile eggs were obtained. Further loss in the number of normal spermatozoa was observed following the freezing-thawing process. Of the two investigated freezing-thawing methods, the rapid rate (60 C/min) appeared less detrimental to spermatozoa morphology and their ability to fertilize the ovum than the "slow" rate. Also the number of sperm holes appearing in the inner perivitelline layer and the number of spermatozoa trapped in the outer perivitelline layer of the ovum was higher after the rapid than the 'slow' freezing-thawing procedure. Nevertheless, both rates did not yield any fertile eggs.     

视频荧光光谱图像技术鉴别复印纸

为建立使用视频荧光光谱图像技术检验不同品牌、型号复印纸的分析方法,实现对复印纸的检验和鉴别,收集了不同品牌和型号的静电复印纸20种,利用VSC6000视频光谱比较仪对其进行检验,获得样本的荧光图像,结合图像处理技术对荧光形态、荧光强度等进行可视化和量化分析。依据紫外光、强光源工作条件下纸张样品的视频荧光现象的形态学特征可对纸张进行初步鉴别;通过绘制视频荧光图像的等高线分布图可实现对纸张样品的可视化分析;计算荧光图像亮度值L可对纸张样品进行量化检验。研究结果表明,视频荧光光谱图像技术能够对复印纸进行准确、有效的检验,该方法适用于文件物质材料检验,为纸张的鉴别、比对和同一认定提供了一种实用方法。     

基于符号熵特征提取方法的特种品质鸡蛋近红外光谱鉴别分析

特种品质鸡蛋的快速、无损、准确鉴别是目前急需解决的问题。提出利用符号熵特征提取方法进行特种品质鸡蛋的近红外光谱鉴别。选择普通鸡蛋、柴鸡蛋、富硒蛋和富锌蛋作为研究对象,测得其在12 000～4 000cm-1范围内近红外漫反射光谱,基于符号聚合近似算法进行符号化表示,提取符号光谱序列的符号熵作为特征向量,然后设计纠错编码支持向量机多类分类器对光谱进行定性鉴别。算法参数变化时,符号熵特征表现出很好的鲁棒性,最高识别率达100%。研究结果表明利用近红外方法鉴定特种品质鸡蛋是可行的,同时符号熵可作为一种新的近红外光谱特征提取方法。     

Search for the invisible decay of neutrons with KamLAND

The Kamioka Liquid scintillator Anti-Neutrino Detector is used in a search for single neutron or two-neutron intranuclear disappearance that would produce holes in the -shell energy level of (12)C nuclei. Such holes could be created as a result of nucleon decay into invisible modes (inv), e.g., n--> 3v or nn--> 2v. The deexcitation of the corresponding daughter nucleus results in a sequence of space and time-correlated events observable in the liquid scintillator detector. We report on new limits for one- and two-neutron disappearance: tau(n--> inv) > 5.8 x 10(29) years and tau (nn--> inv) > 1.4 x 10(30) years at 90% C.L. These results represent an improvement of factors of approximately 3 and >10(4) and over previous experiments.     

Some attributes of cold hardiness of the gregarious ectoparasitoid Colpoclypeus florus (Hymenoptera: Eulophidae)

Cold hardiness of diapause and non-diapause larvae of the parasitoid Colpoclypeus florus Walker (Hymenoptera: Eulophidae) was examined in the laboratory. Mean supercooling point (SCP) for diapausing larvae was -26.7 degree C and for non-diapausing larvae immediately after their larval development, was -16.2 degree C. Mean SCP for non-diapause larvae at the prepupa stage was -19.1 degree C. A short period of acclimation (1 week at 5 degree C) had no influence on the mean SCPs of both diapause and non-diapause larvae. Pre-freeze mortality for diapause and non-diapause larvae was also studied. A constant exposure of diapause larvae to -6 degree C resulted in high mortality (70.7 percent) after a period of 40 days. In contrast, 6 days at -6 degree C were sufficient to cause the same level of mortality in non-diapause larvae. After exposure of 15 days at -9 degree C, mortality for non-diapause larvae was 70 percent, whereas after 20 days at the same temperature mortality of diapause larvae was 25 percent. The importance of these findings for the cryobiology of C. florus is discussed.     

Use of light, scanning electron microscopy and bioassays to evaluate parasitism by entomopathogenic fungi of the red scale insect of palms (Phoenicococcus marlatti Ckll., 1899)

We have evaluated the parasitism of the red scale insect of the date palm (Phoenicococcus marlatti) by entomopathogenic fungi, using light microscopy (LM), scanning electron microscopy (SEM) and low temperature scanning electron microscopy (LTSEM). Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium cf. psalliotae, were inoculated directly on the scale insects or on insect infested plant material. We found that L. dimorphum and L. cf. psalliotae developed on plant material and on scale insects, making infection structures. B. bassiana was a bad colonizer of date palm leaves (Phoenix dactylifera L.) and did not parasite the scale insects.