ABSTRACT
In cases of suspicious death, various postmortem changes and the insect species infesting the corpse have been employed to compute the time of death. Recently, chemical analysis of the life stages of insects infesting the corpse has revealed poisons and drugs consumed for suicide in the body of the deceased. This new area can provide proof of the cause of death. It is recommended that whether or not there is suitable tissue samples for the analysis, the developmental stages of insects thriving on the carcass should always be collected as exhibits for analysis.
Subject(s)
Cause of Death , Diptera/analysis , Forensic Medicine/methods , Myiasis , Adult , Animals , Diptera/growth & development , Female , Humans , Malathion/analysis , Male , Middle Aged , Phenobarbital/analysis , Suicide , Time FactorsABSTRACT
Diverse samples were examined at a site of water-bird mortality, caused by Clostridium botulinum type C toxin in southern Moravia (Czechoslovakia). The toxin was detected in high concentrations in mute swan (Cygnus olor) carcasses (less than or equal to 1 x 10(6) LD50/g) as well as in necrophagous larvae and pupae of the blow flies Lucilia sericata and Calliphora vomitoria (less than or equal to 1 x 10(5) LD50/g) collected from them. It was detected in lower concentrations (less than or equal to 1 x 10(3) LD50/g) in other invertebrates (ptychopterid fly larvae, leeches, sow-bugs) associated with these carcasses, and occasionally in water samples (8 LD50/ml) close to the carrion. The toxin was not detected in the samples of water, mud or invertebrates collected at a distance greater than or equal to 5 m from the carcasses. The toxin-bearing larvae of L. sericata and C. vomitoria, containing 80,000 LD50/g of type C toxin, were exposed in the mud at the study site for 131 days from November to March. Although the toxin activity decreased 25-fold and 40-fold in the two samples of maggots exposed during this period, it remained very high (less than or equal to 3,200 LD50/g). Birds ingesting a relatively low number of these toxic larvae (or pupae) in the spring could receive a lethal dose of the toxin.
Subject(s)
Bird Diseases/etiology , Botulinum Toxins/analysis , Botulism/veterinary , Diptera/analysis , Animals , Birds , Botulism/etiology , Disease Vectors , Larva/analysis , SeasonsABSTRACT
We have discovered and characterized a novel coagulation factor Xa inhibitor from the salivary gland of the black fly, Simulium vittatum. Salivary glands were surgically dissected from the flies and a crude salivary gland extract was tested for inhibition of a number of coagulation assays. The gland extract inhibited both thrombin and factor Xa. To purify further the factor Xa inhibitor, a factor Xa affinity column was utilized. Final purification of the black fly factor Xa inhibitor was achieved by reverse-phase C8 microbore high pressure liquid chromatography. Inhibition of factor Xa was nearly stoichiometric by the purified inhibitor with no inhibitor of thrombin detected. SDS-polyacrylamide gel electrophoresis indicated the inhibitor had a molecular weight of 18,000 and sequence analysis of the inhibitor revealed a blocked amino terminus. These data indicate that the blood-sucking black fly has evolved a highly potent inhibitor of mammalian coagulation factor Xa to disrupt its host normal hemostatic clotting mechanisms.
Subject(s)
Antithrombin III/isolation & purification , Diptera/analysis , Factor Xa Inhibitors , Amino Acids/analysis , Animals , Antithrombin III/chemistry , Chromatography, Affinity , Chromatography, High Pressure Liquid , Molecular Weight , Salivary Glands/chemistryABSTRACT
The cuticle proteins of the insect Dacus oleae have been isolated by extraction with a solution of 7 M urea. The affinity properties of cuticle proteins, isolated from the third instar larvae (L3DCPs 1-7), to chitin have been studied. Purified cuticle antigens were polymerized by glutaraldehyde and used for raising antibodies. The developmental appearance of the cuticle proteins has been studied by two-dimensional electrophoresis.
Subject(s)
Diptera/growth & development , Insect Proteins , Proteins/isolation & purification , Animals , Antibodies , Chitin , Chromatography, Affinity , Diptera/analysis , Electrophoresis, Gel, Two-Dimensional , Molecular Weight , Solubility , UreaABSTRACT
Thin-layer chromatography of the cuticular lipids of horse flies from Oklahoma revealed that hydrocarbon was the major lipid class present. The hydrocarbon fraction was composed of n-alkanes and methyl branched alkanes with only a small amount of alkenes present. Gas chromatography of the isolated cuticular hydrocarbons from a single species, Tabanus abactor Philip, showed no major differences in the profiles between individuals or between the sexes. Analysis of extracts of fresh, frozen, and pinned specimens yielded nearly identical hydrocarbon profiles. Profiles of several species were examined and found to be unique for each. Three species with similar morphological characteristics and similar geographical ranges. Tabanus abdominalis F., T. limbatinevris Macquart, and T. sulcifrons Macquart, were differentiated easily by comparison of the hydrocarbon profiles.
Subject(s)
Diptera/isolation & purification , Hydrocarbons/analysis , Animals , Chromatography, Gas , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Densitometry , Diptera/analysis , Female , MaleABSTRACT
A hypertrehalosaemic neuropeptide from the corpora cardiaca of the blowfly Phormia terraenovae has been isolated by reversed-phase h.p.l.c., and its primary structure was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The octapeptide has the sequence pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 and is clearly defined as a novel member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of peptides. It is the first charged member of this family to be found. The synthetic peptide causes an increase in the haemolymph carbohydrate concentration in a dose-dependent fashion in blowflies and therefore is named 'Phormia terraenovae hypertrehalosaemic hormone' (Pht-HrTH). In addition, receptors in the fat-body of the American cockroach (Periplaneta americana) recognize the peptide, resulting in carbohydrate elevation in the blood. However, fat-body receptors of the migratory locust (Locusta migratoria) do not recognize this charged molecule, and thus no lipid mobilization is observed in this species.
Subject(s)
Diptera/analysis , Insect Hormones/isolation & purification , Neurosecretory Systems/analysis , Oligopeptides/isolation & purification , Oligopeptides/metabolism , Amino Acid Sequence , Amino Acids/analysis , Animals , Chromatography, High Pressure Liquid , Molecular Sequence Data , Pyrrolidonecarboxylic Acid/analogs & derivativesABSTRACT
The two major components of the acidic glycolipid fraction from the pupae of Calliphora vicina were isolated using high-performance liquid chromatography. The acidic moiety was identified as glucuronic acid by beta-glucuronidase cleavage and gas chromatographic analysis as the pentafluoropropionyl derivative. The structures of the carbohydrate moiety were elucidated by peracetylation, methylation, exoglycosidase cleavage, fast-atom-bombardment mass spectrometric and 1H-nuclear magnetic resonance spectroscopic analysis. The only difference between the two hexasaccharide variants was the presence, in one of them, of a between the two hexasaccharide variants was the presence, in one of them, of a phosphoethanolamine (AeP) sidechain on the third sugar of the sequence, i.e. N-acetylglucosamine. The composition of the ceramide moiety was dominated by a C20:0 fatty acid (arachidic acid) and a C14:1 sphingoid base (tetradecasphing-4-enine). The chemical structures of the two insect acidic glycosphingolipids were determined to be: GlcA(beta 1-3)Gal-(beta 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man (beta 1-4)Glc(beta 1-1)Cer; GlcA(beta 1-3)Gal(beta 1-3)GalNAc(beta 1-4)[2AeP-6]-GlcNAc(beta 1-3) Man(beta 1-4)Glc(beta 1-1)Cer. Such glucuronic-acid-containing insect glycosphingolipids have been given the generic name arthrosides, with the implied synonymity to the gangliosides.
Subject(s)
Acetylglucosamine/analysis , Ceramides/analysis , Diptera/analysis , Ethanolamines/analysis , Glucosamine/analogs & derivatives , Glucuronates/analysis , Glycosphingolipids/isolation & purification , Polysaccharides/analysis , Animals , Binding Sites , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, Thin Layer , Glucuronic Acid , Glycoside Hydrolases , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Mass SpectrometryABSTRACT
Toxicological analyses on a putrefied cadaver are sometimes difficult to achieve, due to the absence of blood and/or urine. In this study, morphine and phenobarbital were simultaneously identified and assayed in several tissues of a putrefied cadaver and in the fly larvae of Calliphoridae found on the corpse.
Subject(s)
Cadaver , Diptera/analysis , Morphine/analysis , Phenobarbital/analysis , Adult , Animals , Chromatography, Gas , Chromatography, Liquid , Fluorescence Polarization Immunoassay , Humans , Larva/analysis , MaleABSTRACT
Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.
Subject(s)
Insecta/analysis , Nervous System/analysis , Sodium Channels/analysis , Amino Acid Sequence , Animals , Cockroaches/analysis , Cyclic AMP/pharmacology , Diptera/analysis , Disulfides/metabolism , Glycoproteins/analysis , Grasshoppers/analysis , Immunosorbent Techniques , Molecular Sequence Data , Moths/analysis , Neurons/analysis , Peptide Mapping , Phosphorylation , Protein Kinases/metabolism , Sodium Channels/metabolismABSTRACT
Two novel phosphorus-containing neutral glycosphingolipids of the arthro series were isolated from the blowfly Calliphora vicina Meigen: GalNAc alpha 1----4GalNAc beta 1----(X---- 6)4GlcNAc beta 1----3Man beta 1----4Glc beta 1----1-ceramide and GalNAc beta 1----(X----6)4GlcNAc beta 1----3Man beta 1----4Glc beta 1----1- ceramide (X = -O-P(O)(O-)-OC-H2CH2NH3+). The primary structure of the ceramide pentasaccharide was elucidated de novo using two-dimensional 1H NMR correlation spectroscopy at 500 MHz and multistep relayed coherence transfer spectroscopy at 600 MHz. Localization of the 2'-aminoethyl phosphate substituent was established with the aid of 1H-detected, 31P-edited NMR spectroscopy at 500/202 MHz.
Subject(s)
Diptera/analysis , Glycosphingolipids/isolation & purification , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, Thin Layer , Hydrogen , Magnetic Resonance Spectroscopy/methods , Molecular Sequence Data , Phosphorus , PupaABSTRACT
We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase-sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.
Subject(s)
Calcium/analysis , Extracellular Space/analysis , Microelectrodes , Amoeba/analysis , Animals , Calcium/physiology , Dictyostelium/analysis , Diptera/analysis , Electrophysiology , Extracellular Space/physiology , Pollen/analysis , Seaweed/analysis , Urochordata/analysis , VibrationABSTRACT
Sapecin is a 40-residue peptide containing 6 half-cystine residues. The disulfide structure of sapecin was determined by sequencing cystine-containing peptides obtained by digesting sapecin with thermolysin. Results showed that sapecin has a vortical structure fixed by 3 disulfide bonds between cysteine residues 3 and 30, 16 and 36, and 20 and 38, respectively, and that these disulfide bonds are essential for its antibacterial activity.
Subject(s)
Anti-Infective Agents/analysis , Diptera/analysis , Disulfides/analysis , Insect Hormones/analysis , Insect Proteins , Amino Acid Sequence , Animals , Cells, Cultured , Chromatography, High Pressure Liquid , Cysteine/analysis , Dithiothreitol , Molecular Sequence Data , Solubility , ThermolysinABSTRACT
Toxicological analyses on a putrefied cadaver are sometimes difficult to perform because of the absence of blood and urine. In this study, fly larvae, being living material, are proposed as a new medium of investigation in forensic toxicology. Bromazepam and levomepromazine were identified and assayed in the remains of cerebral tissue, in the clavicle of a putrefied cadaver, and in the fly larvae found on and in the corpse.
Subject(s)
Anti-Anxiety Agents/analysis , Bromazepam/analysis , Cadaver , Cause of Death , Diptera/analysis , Forensic Medicine/methods , Methotrimeprazine/analysis , Aged , Animals , Brain Chemistry , Humans , Larva/analysis , Male , Toxicology/methodsABSTRACT
Specimens of liver were collected from 40 cases in which the cause of death had been determined to be opiate intoxication. Rearings of Calliphora vicina larvae were then promoted on the decomposing liver. A control group of 10 decomposed liver specimens from non-opiate deaths was treated similarly. Analysis of larvae and liver for opiates (morphine) was conducted by radioimmunoassay. Good qualitative and quantitative correlation was observed in both the positive and negative groups. Regression analysis comparing the concentrations of opiates found in the larvae with those found in the liver in the positive group resulted in a correlation of r = 0.790.
Subject(s)
Cadaver , Diptera/analysis , Liver/analysis , Narcotics/poisoning , Animals , Gas Chromatography-Mass Spectrometry , Humans , Larva/analysis , Morphine/analysis , Narcotics/analysis , Radioimmunoassay , Regression AnalysisABSTRACT
Toxicological analyses on a putrefied cadaver are sometimes difficult to achieve because of the absence of blood and urine. In this study, maggots, living material, are proposed as a new medium of investigation in forensic medicine. Five drugs (triazolam, oxazepam, phenobarbital, alimemazine, and clomipramine) were identified and assayed in some tissues of a putrefied cadaver and in the maggots found on and in the body.
Subject(s)
Benzodiazepines/analysis , Cadaver , Clomipramine/analysis , Diptera/analysis , Phenobarbital/analysis , Trimeprazine/analysis , Animals , Humans , Larva/analysis , Male , Middle Aged , Oxazepam/analysis , Postmortem Changes , Triazolam/analysisABSTRACT
A polyclonal antiserum was prepared against an N-terminal modified Cam-HrTH-II (Leu-Asn-Phe-...), one of the members of the large AKH/RPCH peptide family, first isolated from Carausius morosus. The localisation of this peptide was performed by means of immunocytochemical methods in the brain and corpora cardiaca-corpora allata complex of the stick insect, Carausius morosus and the grey fleshfly, Sarcophaga bullata. The distribution patterns of molecules reactive to the Cam-HrTH-II and the Lom-AKH-I antisera in both insect species were compared. In Carausius, both antisera reacted in the same cell bodies. In Sarcophaga, some neurons were stained by both, others only by one of the two antisera. By combining two different antisera, we demonstrated that there are no Lom-AKH-I-like molecules present in Carausius and that there must occur at least three different AKH-like molecules in the brain of Sarcophaga. One is similar to Cam-HrTH-II, the second to Lom-AKH-I and the third is an AKH/RPCH-like peptide, different from Lom-AKH-I and Cam-HrTH-II.
Subject(s)
Diptera/analysis , Insect Hormones/analysis , Insecta/analysis , Oligopeptides , Amino Acid Sequence , Animals , Immunoenzyme Techniques , Molecular Sequence Data , Nervous System/analysisABSTRACT
Tissue of an insect, Lucilia cuprina, fixed conventionally in buffered glutaraldehyde and osmium and embedded in epoxy resin (epon or epon/araldite), provided sections which could readily be labeled with RNAse/gold and wheat germ agglutinin (WGA)/gold. This method offers labeling of tissues with improved contrast and allows the retrospective application of RNAse and WGA labeling to conventionally prepared tissues, without recourse to oxidizing/etching agents.
Subject(s)
Chitin/analysis , Histocytochemistry/methods , RNA/analysis , Ribonucleases , Wheat Germ Agglutinins , Animals , Colloids , Diptera/analysis , Epoxy Resins , Gold , Osmium TetroxideABSTRACT
Neuronal pathways immunoreactive to antisera against the extended-enkephalins, Met-enkephalin-Arg6-Phe7 (Met-7) and Met-enkephalin-Arg6-Gly7-Leu8 (Met-8), have been identified in the brain of the blowfly Calliphora vomitoria. Co-localisation with other enkephalins in certain neurons suggests that a precursor similar to preproenkephalin A exists in insects and that differential enzymatic processing occurs as in vertebrates. Co-localisations of the extended-enkephalin-like peptides with other vertebrate-type peptides, including cholecystokinin and pancreatic polypeptide, also occur. The enkephalinergic pathways are specific, comprising a few groups of highly characteristic neurons and areas of neuropil. Of special interest is the finding that parts of the antennal chemosensory and the optic lobe visual systems contain Met-8 immunoreactive neurons. Within the median neurosecretory cell groups, some of the giant neurons show immunoreactivity to Met-8 and others to both Met-8 and Met-7. Fibres from these cells project to the corpus cardiacum and also to the suboesophageal ganglion, where arborisations occur in the tritocerebral neuropil. Co-localisation studies of these cells have shown that at certain terminals, one particular type of peptide is the dominant neuroregulator, whilst at other terminals, within the same cell, a different co-synthesised peptide predominates. Several groups of lateral neurosecretory cells show clearly defined enkephalinergic pathways, most of which have connections with the central body. The complex patterns of immunoreactivity seen in terminals in the different parts of the central body, suggest an important role for the enkephalin-like peptides in the integration of multimodal sensory inputs. The physiological functions of the extended-enkephalin-like peptides in the brain of Calliphora is still unknown, but the anatomical evidence suggests they may have a role similar to that in mammals, where they are thought to control aspects of feeding behaviour.
Subject(s)
Diptera/analysis , Enkephalin, Methionine/analogs & derivatives , Neurons/analysis , Animals , Brain/cytology , Brain Chemistry , Cross Reactions , Diptera/cytology , Enkephalin, Methionine/analysis , Enkephalin, Methionine/physiology , Female , Immunoenzyme Techniques , Male , Neural Pathways/analysis , Neural Pathways/cytologyABSTRACT
Parity, stage of follicular development, sperm and fructose presence were determined for 6 tabanid species from southwestern Quebec during 2 consecutive years. Females were collected in canopy traps baited with or without carbon dioxide. Based on the presence or absence of host-seeking nulliparous flies (nullipars), Hybomitra epistates, H. nitidifrons nuda, H. sodalis, Tabanus lineola and T. similis were classified as being anautogenous. Hybomitra frontalis was classified as autogenous for its first ovarian cycle. Sperm was found in 88% and fructose was detected in 84% of the 700 specimens dissected. In anautogenous species, sperm and fructose prevalence was higher in pars than in nullipars.