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Species Identification of Necrophagous Insect Eggs Based on Amino Acid Profile Differences Revealed by Direct Analysis in Real Time-High Resolution Mass Spectrometry

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† Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States
‡ John Jay College of Criminal Justice, 524 West 59th Street, New York, New York 10019, United States
*E-mail: [email protected]
Cite this: Anal. Chem. 2017, 89, 14, 7719–7726
Publication Date (Web):June 6, 2017
https://doi.org/10.1021/acs.analchem.7b01708

Copyright © 2017 American Chemical Society. This publication is licensed under these Terms of Use.

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Abstract

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The colonization of decomposing remains by necrophagous insects such as blow flies is of forensic importance because the progression through the various stages of insect development can be correlated to time of death. The ability to infer this information hinges on accurate determination of the fly species that are associated with the entomological evidence collected. This evidence can include eggs, larvae, pupae, and puparial casings. Determination of the egg’s identity is particularly challenging because the eggs of multiple species are morphologically very similar. We report here that the species identity of fly eggs can be determined from their chemical fingerprint signatures acquired by direct analysis in real time high-resolution mass spectrometry (DART-HRMS). Thus, freshly laid eggs were collected and readily distinguished from multiple necrophagous fly species in the Manhattan area of New York City. These species included representatives from the blow fly family (Calliphoridae), specifically Calliphora vicina, Lucilia sericata, L. coeruleiviridis, and Phormia regina species as well as the Phoridae and Sarcophagidae families. Multivariate statistical analysis of their observed DART-HRMS spectra revealed intraspecies similarities and interspecies differences that were the basis of species differentiation. The chemical basis of discrimination was differences in amino acid profiles. This represents the first chemically based fly egg identification approach with applications to forensic entomology. The rapidity of the method makes feasible the generation of a fly egg chemical profile database against which the DART-HRMS spectra of unknown eggs can be screened to rapidly assess species identity without needing to rear the eggs to adulthood.

It has long been recognized that colonization of corpses by various insect types can provide critical information about the circumstances under which a death occurred. (1, 2) In this regard, the utility of “carrion insects” such as those of the order Diptera is a consequence of the fact that: (1) under specific temperature and weather conditions, they exhibit predictable growth rates that can be correlated to time of death or post-mortem interval (PMI), and (2) in general, the predictable order in which insect species arrive (termed succession) is a function of the level of decomposition. (1, 3-6) The ability to infer this crucial information hinges on correct identification of the species associated with any insect-related evidence retrieved at or in the vicinity of the corpse. Members of the insect family Calliphoridae, which include most species of blow flies, are typically the earliest arrivers, often appearing within ∼5–15 min after death. (7) Flies at a distance of up to 1–2 miles are attracted to the corpse via chemical and/or visual cues, and the corpse is used as a food resource for larvae (i.e., maggots) which emerge from hatched eggs. (8, 9) Because the adult flies disperse when the corpse is disturbed, the most valuable entomological evidence which can be retrieved for species identification is that left behind by female flies such as eggs, larvae, or the remnants of other life stages such as puparial casings. (10) However, although each life stage is clearly distinguishable from the others, the same life stage of closely related species are often difficult to tell apart. For example, even to the trained eye, it is almost impossible to differentiate between the eggs of multiple fly species. (11)Figure 1 illustrates the similarities between the eggs of four Calliphoridae species (Lucilia sericata, Phormia regina, Lucilia coeruleiviridis, and Calliphora vicina), and demonstrates that although differences between adult flies are visually more readily apparent, the eggs are not as easily distinguished. Thus, it remains common practice to rear the eggs to adulthood so that the macroscopic features of the adult fly can be used for species identification. This is a time- and resource-intensive process which not only requires expert knowledge of rearing temperatures and optimal incubation periods, but also necessitates that the recovered eggs be viable. (12) Importantly, eggs retrieved from corpses are often suspended in aqueous ethanol on-site, which precludes the possibility of rearing them to adulthood. (13, 14) Identification of larvae and pupae presents similar challenges. Their weights, dimensions, and morphological features can be compared with published standard charts to facilitate species identification and determination of possible age. (15-17) However, this approach can yield inaccurate results not only due to a lack of reliable taxonomic keys, but also because of the common appearance of developmental variations that emerge as a consequence of differences in nutritional exposure or the presence of toxicological contaminants. (18-20) Furthermore, the manner in which death occurred and the conditions of the corpse can greatly alter the size of the larvae, making accurate interpretation of their physical characteristics tenuous.

Figure 1

Figure 1. Images of eggs derived from four blow fly species from the Calliphoridae family, with the inset in each case showing a picture of the adult fly species corresponding to the eggs displayed in each panel. (A) Lucilia sericata eggs and adult fly; (B) Phormia regina eggs and adult fly; (C) L. coeruleiviridis eggs and adult fly; (D) Calliphora vicina eggs and adult fly. The scale provided refers to egg images only and does not reflect adult size.

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For these reasons, alternative species determination methods have been explored. Light and scanning electron microscopy with or without sample staining have been used to distinguish between eggs of different genera. (21) However, it can be quite challenging to differentiate between the eggs of closely related species. (22) Furthermore, identification by this approach relies on the existence of entomological keys to which the images can be compared, and these are often not available for a broad enough range of species. (11) DNA barcoding using a 600 bp fragment of the mitochondrial cytochrome c oxidase gene to assign individuals to species is another method that is being developed. (23) This approach is potentially very powerful because tissue from any available life stage can be used as source material. (24, 25) In addition, insect life stage parts need not be intact, as is required for visual or microscopy-facilitated identification. (24, 26) However, patterns of mitochondrial variability can be confounded by the spread of maternally transmitted bacteria such as Wolbachia that cosegregate with mitochondria. (23, 27) This can result in low species identification success rates due to monophyly of many species at the mitochondrial level (i.e., different species will have identical barcodes). (23, 24) This approach also requires the development of comprehensive DNA barcodes for the hundreds of relevant species, which is time-consuming, labor-intensive, and has yet to be accomplished. (26) An emerging technique for species identification is hydrocarbon profiling. Cuticular hydrocarbon composition varies as a function of age and species, and thus, chemical profiles can be used to discriminate between different species of insects. (3, 28) This approach has been applied successfully to distinguish between different species of African and European honeybees and pine engraver beetles. (29-31) Hydrocarbon profiles of puparial casings have also been shown to correlate to species. (32) In addition, it has been demonstrated that multivariate statistical analysis processing of these profiles can aid in species differentiation and confer an additional level of statistical certainty to the identification. (32)
It would be highly advantageous to be able to rapidly determine the species of entomological evidence representative of the earliest fly life stages, most notably the eggs, because this would circumvent the need for further time-consuming analyses associated with more commonly used conventional species identification methods. Desirable features of such a method would include avoiding the need for the eggs to be reared to adulthood, as well as the sample preparation steps required for microscopic analysis. The ability to directly analyze evidence in the form in which it is prepared at the crime scene would be an added benefit, as would the reporting of species identification confidence levels. Toward the goal of developing an approach that would fulfill the aforementioned requirements, we show here that the chemical profiles of ethanol solutions in which blow fly eggs are stored exhibit egg-derived chemical fingerprints that can be used to identify species. These signatures can be rapidly determined using direct analysis in real time-high resolution mass spectrometry (DART-HRMS), and chemometric processing of this data provides a means whereby species can be identified. We demonstrate proof-of-principle using eggs verified to be derived from four blow fly species (family Calliphoridae): Calliphora vicina (Robineau–Desvoidy), Lucilia sericata (Meigen), L. coeruleiviridis (Macquart), Phormia regina (Meigen), and members of the Phoridae and Sarcophagidae families.

Methods

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Chemical Standards

The following amino acid standards were purchased from Sigma-Aldrich (St. Louis, MO): arginine, glutamine, isoleucine, lysine, proline, and valine. Cysteine, glutamic acid, serine, and tyrosine were acquired from Oakwood Products (Estill, SC). The MALDI matrix 2,5-dihydroxybenzoic acid (DHB) was purchased from Acros Organics (Morris Plains, NJ). The following solvents were purchased from the indicated vendors: acetonitrile, ethanol, and n-butanol (Fisher Scientific, Pittsburgh, PA); acetone (Pharmco-Aaper, Shelbyville, KY); trifluoroacetic acid (Acros Organics, Morris Plains, NJ); and acetic acid (Mallinckrodt, St. Louis, MO). Ninhydrin was purchased from Pierce Chemical (Rockford, IL) and silica gel IB-F thin layer chromatography plates (2.75 × 7.5 cm) were purchased from J.T. Baker (Center Valley, PA).

Photographic Imaging

Adult specimens were obtained from the Forensic Entomology Insect Collection at John Jay College. Adult flies were pinned through the thorax using #3 BioQuip insect pins (Rancho Dominguez, CA) and placed on a Styrofoam platform. Photographs were taken using a Nikon D5500 camera with a Nikon AF-S MicroNikkor 105 mm micro-lens. Egg specimens were obtained by placing 35 g of fresh pork liver into rearing colonies maintained by the Dr. Rosati Lab at John Jay College. Liver was placed into colony cages of L. sericata, P. regina and C. vicina where it remained for 16 h. Deposited eggs were collected. Eggs of L. coeruleiviridis were obtained from a preserved sample derived from a colony maintained during spring 2016. Fresh pork liver (35 g) was placed in the colony cage for approximately 12 h, and eggs were collected and stored in 70% ethanol solution. Blow fly eggs (fresh and preserved) were placed on 35 g of fresh pork liver for imaging purposes. Images were taken using a Zeiss Discovery v8 Stereoscope with a 10x ocular lens, and a Plan S 1.0x FWD 81 mm objective lens set on a magnification of 1×.

Collection and Preservation of Blow Fly Eggs

Stock blow fly colonies were maintained in the entomology lab of Dr. Jennifer Rosati (John Jay College of Criminal Justice, New York, NY). Adult flies were housed in 45 × 45 × 45 cm steel and mesh cages with a 12L:12D diel cycle, 50% humidity, and a temperature of 21 °C. (33) Adult flies were fed milk powder and sugar placed in a 100 mm plastic Petri dish (Fisher Scientific, Pittsburgh, PA) and water ad libitum in a 500 mL Erlenmeyer flask plugged with paper towels to prevent drowning. L. sericata and Sarcophagidae colonies were maintained since 2014; Phoridae colonies were started in 2015, and C. vicina and L. coeruleiviridis colonies began in 2016, all with flies collected from the Manhattan area. P. regina colonies were originally obtained from Dr. Christine Picard at Indiana University-Purdue University (Indianapolis) and subsequently established at John Jay College in 2015. All colonies are augmented annually with wild-type females collected from the Manhattan, NY area with traps baited with pork liver. For the collection of the fresh eggs used in these experiments, pork liver was used as an oviposition medium and was placed inside each colony cage until sufficient eggs (approximately 100) were collected. The eggs were then removed from the tissue and were suspended in 70% aqueous ethanol storage solutions. Approximately 10–50 eggs of a given species were placed into a vial containing 3 mL of solution. Five replicate vials were prepared for each of the four species of blow fly: C. vicina, L. sericata, L. coeruleiviridis, P. regina, and members of the Phoridae and Sarcophagidae families. The samples were stored at 4 °C until analysis.

DART-HRMS Experiments

Mass spectral data were acquired using a DART-SVP ion source (IonSense, Saugus, MA) attached to a JEOL AccuTOF mass spectrometer (JEOL USA, Peabody, MA) in positive-ion mode. The DART ion source grid voltage was 50 V, and the gas heater temperature was set to 350 °C. Mass spectrometer settings were: ring lens voltage, 5 V; orifice 1 voltage, 20 V; orifice 2 voltage, 5 V; and peak voltage, 400 V. The helium flow rate of the DART ion source was 2.0 L/min. Spectra were collected at 1 spectrum per s over the mass range m/z 40–800. The resolving power of the mass spectrometer was 6000 FWHM. Mass calibration was performed using polyethylene glycol (PEG 600).
Ethanol suspensions of fly egg masses that were created by the addition of eggs to the aqueous ethanol storage solutions were analyzed by dipping the closed end of a melting point capillary tube into the solution and presenting the coated surface to the open-air space between the mass spectrometer inlet and ion source. A minimum of three replicates of each sample were analyzed and averaged together, and five samples of each species were tested to ensure a comprehensive data set. The chemical standards, amino acids, and calibrants, all of which were solids, were also analyzed by dipping the closed end of a melting point capillary tube into the powder and presenting it to the ion source.
In-source collision-induced dissociation (CID) by DART-HRMS was performed on ethanol egg suspension samples and standard solutions using function switching, where spectra are acquired with a predefined set of increasing orifice 1 voltages within a single MS experiment. In these analyses, the orifice 1 voltage was varied from 20, 30, 60, and 90 V, which caused increasing levels of fragmentation.
Data processing, including calibration, background subtraction, and peak centroiding, were performed using TSSPro3 software (Schrader Analytical Laboratories, Detroit, MI). Mass spectral analysis, elemental composition, and linear discriminant analysis (LDA) were conducted using Mass Mountaineer (Mass-spec-software.com, RBC Software, Portsmouth, NH).

Multivariate Statistical Analysis of DART-HRMS-Derived Chemical Fingerprints

Multivariate statistical analysis processing was accomplished through linear discriminant analysis (LDA). The raw mass spectral data were used to generate principal components (PCs) based on peaks within a 7 millimass unit tolerance and above a threshold of 5% relative abundance. The LDA plot was created from the first three PCs, and evaluation of the classification system was performed by leave-one-out cross-validation (LOOCV). In this validation technique, each sample is excluded individually from the training set, which is then rebuilt, and then the excluded sample is tested as an unknown to determine the class to which it is assigned by the model. This process is repeated for each data point, and the strength of the model is assessed by the accuracy of the predicted classification assigned to each sample.

Amino Acid Identification by MALDI-MS/MS Analysis of Eggs

Matrix assisted laser desorption ionization-high resolution mass spectrometry (MALDI-HRMS) analyses were performed using a JMS-S3000 SpiralTOF MALDI TOF/TOF mass spectrometer (JEOL USA, Peabody, MA). Mass spectral data were recorded in positive spiral mode, and spectra were obtained in the range of m/z 50–1000 at a sampling interval of 0.5 ns. The laser frequency was set to 250 Hz. Several instrument parameters such as detector power, delay time, and laser intensity were optimized for each sample analysis. The delay time was set between 10 and 20 ns; the detector power was generally between 60 and 70%, and the laser intensity was ∼85%. Data processing was performed using msTornado and msMicroImager (JEOL Ltd., Akishima, Tokyo, Japan) and included centroiding and peak selection as well as the compilation of ion image renders. Initial analyses of the amino acid standards and egg extract solutions were performed in spiral mode to determine the elemental composition of the amino acids present. Next, individual ions were selected for further MS/MS analysis to confirm the presence of individual amino acids.
The matrix solution used for analyses was comprised of 10 mg/mL DHB in 1:1 water:acetonitrile with 1% trifluoroacetic acid (v/v). Authentic amino acid standards were first analyzed for later comparison to egg extract analysis results. Standard solutions comprised of 5 mg of each amino acid in 0.5 mL of 1:1 water:acetonitrile with 1% trifluoroacetic acid (v/v) were prepared. For MALDI analysis, a mixture of 1 μL of amino acid standard solution in 10 μL of matrix solution was applied to a stainless steel target plate.
To detect and confirm the presence of amino acids in eggs, approximately 0.05 g of eggs was transferred to a 2.0 mL Eppendorf tube and ground into a paste using a metal spatula. Acetonitrile (1 mL) was added. The mixture was sonicated using a Bransonic B-3 Ultrasonic Cleaner (Branson Ultrasonics, Danbury, CT) for 30 min and then centrifuged. The supernatant was removed with a pipet and transferred to an Eppendorf tube, which was left uncapped for approximately 24 h to allow the solvent to evaporate completely. The resulting residue was then resuspended in 100 μL of acetonitrile. This solution (5 μL) was then analyzed by MALDI-SpiralTOF HRMS by first adding 6 μL of the matrix solution and then transferring 0.5 μL of the resulting mixture onto a stainless steel target plate. The external calibrant used for these experiments was prepared by adding 1 μL of PEG 200 to 10 μL of matrix solution. The amino acid standards, egg samples, and calibrant were applied to stainless steel target plates (0.5 μL) and allowed to air-dry before MALDI-HRMS analysis.

Amino Acid Identification by Thin Layer Chromatography (TLC)

Because alanine and glycine have masses below 100 Da, it was not possible to confirm their presence by MS/MS using our MALDI-HRMS instrument. Therefore, to confirm the presence of these amino acids in the fly eggs, analysis by TLC was performed. Eggs (∼0.1 g) were placed into an Eppendorf tube and ground into a paste using a metal spatula. To this was added 500 μL of the TLC elution solvent which was comprised of n-butanol, acetic acid, and water (3:1:1 v/v). The egg sample was then sonicated for 20 min. Glycine and alanine standard solutions were prepared by adding 5 mg of the amino acid to 1 mL of the TLC elution solvent. The standards and egg solutions were then spotted onto a TLC plate, which was developed in the TLC elution solvent for approximately 1.5 h. After development was complete, the plate was removed from the TLC chamber and allowed to air-dry. A ninhydrin stain comprised of 0.1 g of ninhydrin dissolved in a solution of 0.5 mL acetic acid and 100 mL acetone was applied to the entire plate using a plastic spray bottle (CVS, Woonsocket, RI). The plate was again air-dried before it was placed in a 100 °C oven for 3 min. During this time, colors indicative of the presence of each individual amino acid appeared. The Rf values of the spots representative of the amino acid standards and the amino acids in the eggs were calculated.

Results

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Necrophagous Fly Egg Species Identification and Differentiation

DART-HRMS analyses of ethanol samples in which blow fly eggs were suspended were performed in positive ion mode at 350 °C. The results for the six fly species C. vicina, L. coeruleiviridis, L. sericata, P. regina, Phoridae, and Sarcophagidae are shown in Figure 2, and the related mass measurement data, including measured masses and relative peak abundances, are listed in Supporting Information Table S1. Visual inspection of the spectra showed that each species exhibited a unique chemical fingerprint. The spectra displayed between 21 and 90 peaks above a threshold of 2% relative abundance depending on the species. L. coeruleiviridis had the greatest number of peaks, and Sarcophagidae had the least.

Figure 2

Figure 2. Results of DART-HRMS analyses of aqueous ethanol suspensions of six species of fly eggs: (a) C. vicina, (b) L. coeruleiviridis, (c) L. sericata, (d) P. regina, (e) Phoridae spp., and (f) Sarcophagidae spp. All analyses were performed in positive ion mode at 350 °C. Each spectrum represents an average of three analyses. The spectra generated for each species are unique and can be used as fingerprints for the identification of individual species.

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Although the chemical profile of each species was unique, some peaks were common to all species, including nominal m/z 93 and 100. Others were observed in most of the spectra such as m/z 90, which appeared in the spectra of every species except for Phoridae, and nominal m/z 72 and 115, which were observed in the spectra of all species except Sarcophagidae. A number of peaks were only seen in a small subset of spectra. For example, m/z 76 was observed only in L. coeruleiviridis, C. vicina, and P. regina; m/z 288 and 289 were detected only in L. coeruleiviridis and Sarcophagidae; and m/z 246 was seen only in C. vicina. Nominal m/z 271 was detected only in L. sericata and P. regina. There were a handful of peaks that were observed only in the four species C. vicina, L. coeruleiviridis, L. sericata, and P. regina. These included nominal m/z 311. Additionally, peaks such as m/z 120, 130, and 146 appeared only in C. vicina, L. coeruleiviridis, and P. regina.
From the high-resolution data, it was possible to make tentative structural assignments for several of the detected masses. A peak at nominal m/z 93 was observed in the spectra of all 6 ethanol egg suspensions and corresponded to the formula C4H12O2 + H+ or the protonated dimer of ethanol. Many of the observed peaks had formulas consistent with those of amino acids. The peak at nominal m/z 116, which was detected in the mass spectral profiles of C. vicina, L. coeruleiviridis, P. regina, L. sericata, and Sarcophagidae, corresponded to the formula C5H9NO2 + H+ and was tentatively identified as proline. A peak at m/z 90 corresponding to C3H7NO2 + H+ was detected in the spectra of all of the species except Phoridae, and was assigned to alanine. Protonated forms of the isomeric amino acids isoleucine/leucine were also believed to be present in all of the ethanol egg suspensions except Phoridae, as a peak at nominal m/z 132 was observed. The peak at m/z 118 that was detected in the spectra of C. vicina, L. coeruleiviridis, P. regina, L. sericata, and Phoridae was assigned to protonated valine. Glycine (m/z 76), glutamic acid (m/z 148.08), phenylalanine (m/z 166), histidine (m/z 156), methionine (m/z 150), serine (m/z 106), threonine (m/z 120), and tyrosine (m/z 182) were all tentatively identified in the mass spectra of C. vicina, L. coeruleiviridis, and P. regina. Glutamic acid was observed at m/z 148 in C. vicina, L. coeruleiviridis, and P. regina. Glutamine and tryptophan were detected only in the ethanol suspensions of P. regina at masses m/z 147.08 and 205, respectively. The amino acid lysine was tentatively identified in the spectra representing L. coeruleiviridis and P. regina as indicated by the presence of a peak at m/z 147.11. Masses consistent with the presence of the protonated forms of the amino acids arginine, asparagine, aspartic acid, and cysteine at nominal m/z 175, 133, 134, and 122 respectively, were not detected in the spectra of any of the ethanol suspensions.

Confirmation of the Presence of Tentatively Assigned Biomarkers and Amino Acids by DART-HRMS In-Source Collision-Induced Dissociation

The presence of specific compounds can be confirmed by DART-HRMS through comparison of the mass spectral fragmentation pattern observed under in-source CID conditions of an analyzed sample to that of an authentic chemical standard that has been analyzed in a similar manner. (34) A low orifice 1 voltage (e.g., 20 V) produces a spectrum containing unfragmented protonated precursor molecules. By increasing this voltage, fragmentation occurs, and the pattern observed can then serve as a unique fingerprint for a given compound. We used this technique to confirm our tentative structural assignments. To begin these analyses, the optimal orifice 1 voltage (i.e., one which resulted in fragmentation while retaining the protonated precursor) was determined. It was found to be 30 V for alanine, glutamine, tryptophan, tyrosine, and valine, and 60 V for all of the other amino acids. Next, each egg suspension and standard was analyzed by DART-HRMS at this new orifice 1 voltage, and the spectra of the samples were compared to those of the authentic chemical standards analyzed under similar conditions.
The presence of all 15 of the amino acids detected in P. regina under soft ionization DART-HRMS conditions was confirmed by in-source CID. A representative example of the type of data obtained from these experiments is presented in Figure 3 and Supporting Information Figures S1–S14 for P. regina. The head-to-tail plot shown for the amino acid proline in Figure 3 is representative, while similar plots corresponding to the other amino acids are presented in Supporting Information Figures S1–S14. In Figure 3, the top spectrum shows the ethanol suspension of P. regina eggs analyzed at 60 V, and the bottom spectrum is that of the proline standard analyzed under the same conditions. By this approach, the presence of proline in the eggs was confirmed through detection of not only the protonated precursor but also by detection of all of the fragment peaks that were seen for the proline standard. Thus, the fragment peaks observed at nominal m/z 70, 71, 116, and 117 from the proline standard were also observed in the spectrum for P. regina. By performing similar experiments with the other five species of fly eggs, the presence of various amino acids was confirmed (Supporting Information S1–S14). The profile of amino acids identified in each of the analyzed egg species is presented in Table 1.

Figure 3

Figure 3. Head-to-tail plot showing the results of DART-HRMS in-source collision-induced dissociation experiments performed to confirm the presence of proline in P. regina eggs. The top spectrum is of the 60 V experiment of the egg sample, and the bottom spectrum is that of the amino acid analyzed at the same voltage. The presence in the egg spectrum of the peaks that appear in the spectrum of the standard indicate that the proline is present in the blow fly eggs. Both the top and bottom spectra represent an average of three analyses.

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Table 1. Amino Acids Detected in Ethanol Suspensions of Six Species of Fly Egg Samples. Their presence was confirmed by comparing the fragmentation patterns of the in-source CID spectra of the egg suspensions to those of the authentic standards acquired under identical conditions.
amino acid C. vicina L. coeruleiviridis L. sericata P. regina Phoridae spp. Sarcophagidae spp.
alanine x x x x   x
arginine            
asparagine            
aspartic acid            
cysteine            
glutamine       x    
glutamic acid x x   x    
glycine x x   x    
histidine x x   x    
isoleucine/leucine x x x x   x
lysine   x   x    
methionine x x   x    
phenylalanine x x   x    
proline x x x x   x
serine x x   x    
threonine x x   x    
tryptophan       x    
tyrosine x x   x    
valine x x x x x  

Independent Confirmation of the Presence of Amino Acids in Necrophagous Fly Eggs by MALDI-SpiralTOF HRMS

To confirm the DART-HRMS-facilitated amino acid assignments using an alternative method, samples were also analyzed by MALDI-MS/MS. As an example, the spectra obtained for P. regina eggs are presented in Supporting Information Figures S15–S29. Panel a of each image represents the results of the analysis of the egg sample, and panel b shows that of the authentic standard. By MALDI-SpiralTOF HRMS, high-resolution masses consistent with the amino acids arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, isoleucine/leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were observed. The presence of alanine and glycine could not be confirmed by MALDI-MS/MS because their molecular weights are below 100 Da, the threshold for detection by the MALDI-SpiralTOF method. Of the subset of amino acids whose presence was confirmed by MALDI-SpiralTOF HRMS, arginine, asparagine, and aspartic acid were not detected in our DART-HRMS analysis of the blow fly egg ethanol suspensions. However, authentic standards of these amino acids were readily detected by DART-HRMS when analyzed individually.

Confirmation of the Presence of Amino Acids in Necrophagous Fly Eggs by TLC

While the presence of most of the amino acids in the fly eggs could be confirmed by DART- or MALDI-SpiralTOF HRMS, the presence of alanine and glycine was confirmed by TLC analysis of an egg paste extract (Supporting Information Figure S30). The Rf values of the spots observed for the alanine and glycine standards were 0.430 and 0.342 respectively. Six spots were observed in the TLC analysis of the egg sample with Rf values of 0.193, 0.325, 0.430, 0.553, 0.596, and 0.640. Spots with similar Rf values were observed in the analysis of the egg samples. The bands corresponding to alanine and glycine produce characteristic deep red and pale yellow colors, respectively, when stained with ninhydrin, and these same colors were observed for the TLC spots with the Rf values corresponding to glycine and alanine.

Multivariate Statistical Analysis of DART-HRMS Mass Spectral Fingerprints Enabled Species-Level Differentiation

The results of the DART-HRMS analyses of fly eggs revealed intraspecies similarities and interspecies differences, which could in principle be exploited for species identification purposes. To test this hypothesis, the DART-HRMS data were subjected to multivariate statistical analysis. The training set was comprised of 90 spectra in total. Linear discriminant analysis (LDA) was performed using the 15 masses shown in Table 2. These m/z values represented peaks that most species had in common (e.g. nominal m/z 90, 116, and 132 that were observed in all species except Phoridae, m/z 118 and 171 that were detected in all species except Sarcophagidae, and m/z 94 and 100 which were detected in all species); m/z 146 and 166 observed in C. vicina, L. coeruleiviridis and P. regina; m/z 127 detected in L. coeruleiviridis, L. sericata and Phoridae; m/z 147 detected in L. coeruleiviridis and P. regina; m/z 191 only detected in L. coeruleiviridis and L. sericata; m/z 255 and 311 seen in C. vicina, L. coeruleiviridis, L. sericata, and P. regina; and m/z 324 only observed in Phoridae). The leave-one-out cross-validation (LOOCV) was 84%, and 93% of the variance was accounted for by three principal components. Figure 4 shows two orientations of the LDA plot. Clustering of the data points representing the same species was observed.

Figure 4

Figure 4. Two orientations of the results of the linear discriminant analysis (LDA) plot derived from a subset of the m/z values observed in the mass spectra of ethanol suspensions of fly eggs representing the six species indicated. The data set included 90 mass spectral replicates, and 15 feature masses were used (listed in Table 2). These included some that were common to all species and others that were unique to individual species. Three principal components accounted for 93% of the variance, and the leave-one-out cross-validation (LOOCV) was 84%. The plot shows tight clustering of the data points representing samples from the same species, and clear separation between those of different species.

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Table 2. Feature Masses Used for the LDA Plot Presented in Figure 4
90.0557 127.1124 171.1347
94.0953 132.0983 191.1561
100.0734 146.0794 255.2207
116.0669 147.1076 311.2935
118.0840 166.0790 324.2552

Discussion

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The forensic value of juvenile life stages of forensically important fly families such as the eggs would be dramatically enhanced if their species identity could be more readily determined. We demonstrate here that the identification of species belonging to the Calliphoridae, Sarcophagidae, and Phoridae families can be rapidly accomplished through chemometric processing of DART-HRMS-derived chemical fingerprints of ethanol suspensions of eggs. Importantly, the DART-HRMS data is generated from samples of the type typically generated in the field (i.e., suspensions of eggs in aqueous ethanol). Because the ethanol samples are analyzed directly, none of the sample pretreatment steps that are essential for species identification by conventional methods are required. Our mass spectral analyses revealed a number of interesting trends, including: (1) The ethanol suspension of each species exhibited a chemical fingerprint that was remarkably consistent from sample to sample. (2) Chemical fingerprints differed between species, as illustrated by visual inspection of their mass spectra. (3) A subset of peaks was observed only in the mass spectra of C. vicina, L. coeruleiviridis, L. sericata, and P. regina, indicating the ability to distinguish between families as well as species. (4) The intraspecies similarities and interspecies differences of the chemical fingerprints were primarily a consequence of the variations in amino acid profiles of the ethanol suspensions. (5) Cysteine was not detected in any of the ethanol suspensions or the fly paste extracts. (6) DART-HRMS analysis of the ethanol suspension was superior to MALDI-HRMS analysis in that the former revealed a broader range of molecules, including amino acids. Detection of amino acids by MALDI-HRMS was only possible using concentrated extracts made from egg paste. The amino acids arginine, asparagine, and aspartic acid were absent in the DART-HRMS spectra of all six species of fly eggs despite the fact that the chemical standards were easily detected when analyzed by this method. This implies that our inability to observe these compounds in the ethanol whole egg suspensions may be that their levels were too low or because they did not leach into the ethanol/water solution from the eggs. This conclusion is supported by the fact that these amino acids were also not detected in MALDI-SpiralTOF HRMS analyses of the ethanol egg suspensions, but were observed only in samples prepared from macerated eggs (Supporting Information Figures S14–S16). The significance of the absence of cysteine in any of the egg samples is unknown.
It was the consistency of the intraspecies similarities and interspecies differences of the DART-HRMS fingerprints that enabled differentiation and species identification by chemometric processing of the DART-HRMS data. This is illustrated in the LDA plot. An implication of this result is that a DART-HRMS database of fly egg chemical signatures can be used to screen DART-HRMS chemical profiles of fly egg ethanol suspensions collected in the field, and that the results can be used for rapid species identification. To accomplish this, it will be necessary to acquire a comprehensive data set of DART-HRMS spectra representing multiple blow fly and other forensically relevant fly species. However, the speed with which the analyses can be performed (i.e., ∼3 s per analysis) makes the generation of the multiple replicates required quite feasible. Furthermore, the data can be readily expanded to include an even larger number of species. The approach to species determination that is described here provides solutions to some of the challenges of more conventional methods. No sample preparation steps or specialized expertise is required. Furthermore, samples in the form in which they are prepared in the field can be analyzed. The extent to which intraspecies similarities and interspecies differences in chemical fingerprints occurs across the Diptera order is the subject of continuing investigations in our laboratories.

Conclusions

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The technique outlined here is the first demonstration of a rapid chemical fingerprint-based method for species identification of necrophagous fly eggs. The samples were analyzed by DART-HRMS in the form that they are typically collected at a crime scene without any further sample preparation. The analyses revealed that blow fly eggs exhibit species-specific differences in amino acid profiles. The DART-HRMS-chemometric data processing species identification approach circumvents many of the shortcomings of traditional analysis methods, including the time-intensive process of rearing eggs to adulthood to confirm species identity. The chemical profiles revealed intraspecies similarities and interspecies differences that served as the basis for chemometric analysis. The results show the application of DART-HRMS for the generation of unique species-specific chemical fingerprints that can be used to rapidly identify early life stage entomological evidence.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b01708.

  • DART-HRMS in-source CID spectral data of aqueous ethanol egg extracts and amino acid standards, MALDI-MS/MS spectral data of fly eggs, TLC analysis results, and a table of mass measurement data of fly egg spectra (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Justine E. Giffen - Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States
    • Jennifer Y. Rosati - John Jay College of Criminal Justice, 524 West 59th Street, New York, New York 10019, United States
    • Cameron M. Longo - Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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The authors gratefully acknowledge Kristen Fowble for assistance with MALDI-SpiralTOF HRMS experiments, Joey Fragale and Veena Mehta for insect care, Dr. Robert Cody for helpful discussions, and JEOL USA Inc. for the use of the JMS-S3000 SpiralTOF MALDI TOF/TOF mass spectrometer. The financial support of the Research Foundation of SUNY is gratefully acknowledged.

References

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This article references 34 other publications.

  1. 1
    Tabor, K. L.; Brewster, C. C.; Fell, R. D. J. Med. Entomol. 2004, 41, 785– 795 DOI: 10.1603/0022-2585-41.4.785
    Google Scholar
    1
    Analysis of the successional patterns of insects on carrion in southwest Virginia
    Tabor Kimberly L; Brewster Carlyle C; Fell Richard D
    Journal of medical entomology (2004), 41 (4), 785-95 ISSN:0022-2585.
    Studies of carrion-insect succession on domestic pig, Sus scrofa L., were conducted in the spring and summer of 2001 and 2002 in Blacksburg, VA, to identify and analyze the successional patterns of the taxa of forensic importance in southwest Virginia. Forty-seven insect taxa were collected in the spring. These were represented by 11 families (Diptera: Calliphoridae, Sarcophagidae, Muscidae, Sepsidae, Piophilidae; Coleoptera: Staphylinidae, Silphidae, Cleridae, Trogidae, Dermestidae, Histeridae). In the summer, 33 taxa were collected that were represented by all of the families collected in the spring, except Trogidae. The most common flies collected were the calliphorids: Phormia regina (Meigen) and Phaenicia coeruleiviridis (Macquart). The most common beetles were Creophilus maxillosus L. (Staphylinidae), Oiceoptoma noveboracense Forster, Necrophila americana L., Necrodes surinamensis (F.) (Silphidae), Euspilotus assimilis (Paykull), and Hister abbreviatus F. (Histeridae). Occurrence matrices were constructed for the successional patterns of insect taxa during 21 sampling intervals in the spring and 8 intervals in the summer studies. Jackknife estimates (mean+/-95% confidence limits) of overall Jaccard similarity in insect taxa among sampling intervals in the occurrence matrices were 0.213+/-0.081 (spring 2001), 0.194+/-0.043 (summer 2001), 0.257+/-0.068 (spring 2002), and 0.274+/-0.172 (summer 2002). Permutation analyses of the occurrence matrices showed that the patterns of succession of insect taxa were similar between spring 2001 and 2002 (P = 0.001) and between summer 2001 and 2002 (P = 0.007). The successional patterns seem to be typical for the seasonal periods and provide data on baseline fauna for estimating postmortem interval in cases of human death. This study is the first of its kind for southwest Virginia.
    >> More from SciFinder ®
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  2. 2
    Tabor, K. L.; Fell, R. D.; Brewster, C. C. Forensic Sci. Int. 2005, 150, 73– 80 DOI: 10.1016/j.forsciint.2004.06.041
    Google Scholar
    2
    Insect fauna visiting carrion in Southwest Virginia
    Tabor Kimberly L; Fell Richard D; Brewster Carlyle C
    Forensic science international (2005), 150 (1), 73-80 ISSN:0379-0738.
    Successional patterns of insect fauna on pig carcasses were studied in southwest Virginia. The objective was to identify and qualitatively assess the major taxa of forensic importance in this region. Studies were conducted in spring and summer 2001 and 2002, and fall 2002. Over 50 taxa were collected and identified. Phormia regina was the dominant fly species in the spring (>90%) and co-dominant with Phaenicia coeruleiviridis in the summer. Phaenicia sericata, Lucilia illustris, and Calliphora spp. were collected in spring and summer, but less frequently. Eleven species of Sarcophagidae also were collected with Sarcophaga utilis and Helicobia rapax the most common. In the fall, the dominant fly species were Calliphora vomitoria, L. illustris, and P. coeruleiviridis. The primary beetle species collected in spring and summer included three Staphylinidae (Creophilis maxillosus, Platydracus maculosus, and Aleochara lata) and three Silphidae (Oiceoptoma noveboracense, Necrodes surinamensis, and Necrophila americana). No beetles were collected in the fall.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2M3gt1Ogsw%253D%253D&md5=54dcfc1e96361fb00f16b495418b331a
  3. 3
    Pechal, J. L.; Moore, H.; Drijfhout, F.; Benbow, M. E. Forensic Sci. Int. 2014, 245, 65– 71 DOI: 10.1016/j.forsciint.2014.10.019
    Google Scholar
    3
    Hydrocarbon profiles throughout adult Calliphoridae aging: A promising tool for forensic entomology
    Pechal, Jennifer L.; Moore, Hannah; Drijfhout, Falko; Benbow, M. Eric
    Forensic Science International (2014), 245 (), 65-71CODEN: FSINDR; ISSN:0379-0738. (Elsevier Ltd.)
    Blow flies (Diptera: Calliphoridae) are typically the first insects to arrive at human remains and carrion. Predictable succession patterns and known larval development of necrophagous insects on vertebrate remains can assist a forensic entomologist with ests. of a min. post-mortem interval (PMImin) range. However, adult blow flies are infrequently used to est. the PMImin, but rather are used for a confirmation of larval species identification. Cuticular hydrocarbons have demonstrated potential for estg. adult blow fly age, as hydrocarbons are present throughout blow fly development, from egg to adult, and are stable structures. The goal of this study was to identify hydrocarbon profiles assocd. with the adults of a North American native blow fly species, Cochliomyia macellaria (Fabricius) and a North American invasive species, Chrysomya rufifacies (Macquart). Flies were reared at a const. temp. (25 °C), a photoperiod of 14:10 (L:D) (h), and were provided water, sugar and powd. milk ad libitum. Ten adult females from each species were collected at day 1, 5, 10, 20, and 30 post-emergence. Hydrocarbon compds. were extd. and then identified using gas chromatog.-mass spectrometry (GC-MS) anal. A total of 37 and 35 compds. were detected from C. macellaria and Ch. rufifacies, resp. There were 24 and 23 n-alkene and methyl-branched alkane hydrocarbons from C. macellaria and Ch. rufifacies, resp. (10 compds. were shared between species), used for statistical anal. Non-metric multidimensional scaling anal. and permutational multivariate anal. of variance were used to analyze the hydrocarbon profiles with significant differences (P < 0.001) detected among post-emergence age cohorts for each species, and unique hydrocarbon profiles detected as each adult blow fly species aged. This work provides empirical data that serve as a foundation for future research into improving PMImin ests. made by forensic practitioners and potentially increase the use of adult insects during death investigations.
    >> More from SciFinder ®
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  4. 4
    Matuszewski, S.; Bajerlein, D.; Konwerski, S.; Szpila, K. Forensic Sci. Int. 2011, 207, 150– 163 DOI: 10.1016/j.forsciint.2010.09.022
    Google Scholar
    4
    Insect succession and carrion decomposition in selected forests of Central Europe. Part 3: Succession of carrion fauna
    Matuszewski Szymon; Bajerlein Daria; Konwerski Szymon; Szpila Krzysztof
    Forensic science international (2011), 207 (1-3), 150-63 ISSN:.
    The succession of insects on pig carrion was monitored in spring, summer and autumn, in three forest types and 2 years in Western Poland (Central Europe). In most forensically useful taxa, significant differences between seasons, forests and years in time of appearance on carrion were found. The lowest values of appearance time were recorded in summer and the highest in spring. In alder forest insects appeared on carcasses significantly earlier than in pine-oak forest and hornbeam-oak forest. In summer periods of insect presence on carrion were significantly shorter than in spring and autumn. In most taxa no significant effect of forest type or year on length of the presence period was found. In all seasons assemblages of adult taxa were clearly more aggregated than assemblages of larval taxa. Sequence of insects' appearance on carcasses was very similar in different seasons, forests and years. General seasonal models of insect succession on carrion are proposed for forests of Central Europe. Data on appearance time and length of the presence period in particular seasons, forests and years are presented for forensically useful taxa. Implications for methods of PMI estimation (particularly the succession-based method) are discussed.
    >> More from SciFinder ®
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  5. 5
    Matuszewski, S.; Bajerlein, D.; Konwerski, S.; Szpila, K. Forensic Sci. Int. 2010, 194, 85– 93 DOI: 10.1016/j.forsciint.2009.10.016
    Google Scholar
    5
    Insect succession and carrion decomposition in selected forests of Central Europe. Part 1: Pattern and rate of decomposition
    Matuszewski Szymon; Bajerlein Daria; Konwerski Szymon; Szpila Krzysztof
    Forensic science international (2010), 194 (1-3), 85-93 ISSN:.
    Pig carrion decomposition and insect succession were monitored in different seasons and forests of Central Europe. Pattern of decomposition as well as onset, duration and rate of decompositional processes were measured. Pattern of decomposition was the same in almost all cases with putrefaction, active and advanced decay. In the majority of carcasses active decay was driven by larvae of Calliphoridae with a clear seasonal shift in dominant taxa. However, in some spring, alder forest cases active decay was driven by larvae of Necrodes littoralis (Coleoptera: Silphidae). As a rule the mosaic decomposition was observed. In spring a significant delay in onset of all decompositional processes was found. Season significantly affected rate of active decay due to a much higher rate in summer. Decomposition in alder forest proceeded faster than in pine-oak forest and hornbeam-oak forest. Differences between the latter two forests were practically negligible. Implications for forensic entomology are discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3c%252FnsVGntw%253D%253D&md5=aa17ed99481cf74c946b5a51b2e35ed2
  6. 6
    Matuszewski, S.; Bajerlein, D.; Konwerski, S.; Szpila, K. Forensic Sci. Int. 2010, 195, 42– 51 DOI: 10.1016/j.forsciint.2009.11.007
    Google Scholar
    6
    Insect succession and carrion decomposition in selected forests of Central Europe. Part 2: Composition and residency patterns of carrion fauna
    Matuszewski Szymon; Bajerlein Daria; Konwerski Szymon; Szpila Krzysztof
    Forensic science international (2010), 195 (1-3), 42-51 ISSN:.
    The insect fauna of pig carcasses was monitored in different seasons and forests of Western Poland (Central Europe). The composition of carrion fauna and selected features of residency in carrion in adults and larvae of particular taxa were analysed. A total of 131 adult and 36 larval necrophilous taxa were collected. Only 51 adult species and 24 larval taxa were minimally abundant (>or=10 specimens) at least on one carcass. As for the composition of carrion fauna, there were large differences between seasons, but no important differences between forest types. In most species of Diptera, length of the presence period of adults was between 35 and 65% of the sampling interval, while in most species of Coleoptera, it was above 60%. Only in a few species (e.g., Saprinus semistriatus, Necrodes littoralis or Creophilus maxillosus) was the presence period shorter than 35% of the sampling interval. Interestingly, in some adult Coleoptera (e.g., Necrobia violacea) very long presence periods were recorded. In most taxa, the length of the presence period of larvae was between 40 and 65% of the sampling interval. Only Calliphora vomitoria, Phormia regina, Hydrotaea dentipes, N. littoralis and C. maxillosus had shorter presence periods of larvae. As a rule, residency of adults was broken, whereas residency of larvae was unbroken. Moreover, in adults, two distinct residency patterns were observed; with breaks clumped in the final part of the presence period and with breaks evenly distributed inside the presence period. Almost in all taxa, the time of appearance showed the closest relationship to the onset of bloating. The relationship was significant, positive and strong in adults of P. regina, Fannia manicata, Hydrotaea ignava, Stearibia nigriceps, S. semistriatus, N. littoralis and C. maxillosus as well as larvae of P. regina, H. dentipes, H. ignava, S. nigriceps, N. littoralis, Oiceoptoma thoracicum, Thanatophilus sp., C. maxillosus and Philonthus sp. Interestingly, in some forensically significant taxa (e.g., adults of N. violacea or Thanatophilus rugosus), we found no significant relationship between the time of their appearance and the onset of any decompositional process. Implications for the succession-based post-mortem interval (PMI) estimation, determination of the carcass movement and the season of death are discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3c%252FnsFClug%253D%253D&md5=bb02bd16cb2d93a7d3a484dafc8c57cd
  7. 7
    Vasconcelos, S. D.; Soares, T. F.; Costa, D. L. Int. J. Legal Med. 2014, 128, 229– 233 DOI: 10.1007/s00414-013-0936-2
    Google Scholar
    7
    Multiple colonization of a cadaver by insects in an indoor environment: first record of Fannia trimaculata (Diptera: Fanniidae) and Peckia (Peckia) chrysostoma (Sarcophagidae) as colonizers of a human corpse
    Vasconcelos Simao Dias; Soares Thiago Ferreira; Costa Diego Leonel
    International journal of legal medicine (2014), 128 (1), 229-33 ISSN:.
    We describe here a case of multiple colonization of a male cadaver found indoors in the municipality of Jaboatao dos Guararapes, Brazil. The body was colonized by six species of Diptera: Chrysomya albiceps, Chrysomya megacephala, Chrysomya putoria (Calliphoridae), Megaselia scalaris (Phoridae), Fannia trimaculata (Fanniidae), and Peckia (Peckia) chrysostoma (Sarcophagidae). The most abundant species were C. albiceps (65.0 % of all emerged adults) and C. megacephala (18.6 %). The case illustrates the ability of six insect species to simultaneously colonize a corpse in an indoor environment and represents the first collaboration between the forensic police and entomologists in Northeastern Brazil. We provide here the first record of two species, F. trimaculata and Peckia (P.) chrysostoma colonizing a human cadaver. We also report the first case of cadaver colonization by C. putoria and M. scalaris in Northeastern Brazil. Information on the development time of two species, C. albiceps and C. megacephala, were used to discuss the estimation of the post-mortem interval. Considering that the region harbors the highest rates of homicide in Brazil, implications of these findings for the consolidation of forensic entomology in the region are discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c7lt1Sqsg%253D%253D&md5=7f585debc16770e6daf91e3d47227fd9
  8. 8
    Byrd, J. H.; Castner, J. L. Forensic Entomology: The Utility of Arthropods in Legal Investigations; CRC Press: Boca Raton, FL, 2002; p 681.
    Google Scholar
    There is no corresponding record for this reference.
  9. 9
    Greenberg, J. A Natural History of the Chicago Region, 2nd ed.; University of Chicago Press: Chicago, IL, 2002.
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Sukontason, K.; Sribanditmongkol, P.; Ngoen-klan, R.; Klong-klaew, T.; Moophayak, K.; Sukontason, K. L. Parasitol. Res. 2010, 106, 641– 646 DOI: 10.1007/s00436-009-1711-7
    Google Scholar
    10
    Differentiation between Lucilia cuprina and Hemipyrellia ligurriens (Diptera: Calliphoridae) larvae for use in forensic entomology applications
    Sukontason Kom; Sribanditmongkol Pongruk; Ngoen-klan Ratchadawan; Klong-klaew Tunwadee; Moophayak Kittikhun; Sukontason Kabkaew L
    Parasitology research (2010), 106 (3), 641-6 ISSN:.
    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.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3c7hslaktA%253D%253D&md5=b865d0473ab08433e55ee8bcf2703619
  11. 11
    Greenberg, B.; Szyska, M. L. Ann. Entomol. Soc. Am. 1984, 77, 488– 517 DOI: 10.1093/aesa/77.5.488
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Bourel, B.; Callet, B.; Hedouin, V.; Gosset, D. Forensic Sci. Int. 2003, 135, 27– 34 DOI: 10.1016/S0379-0738(03)00157-9
    Google Scholar
    12
    Flies eggs: a new method for the estimation of short-term post-mortem interval?
    Bourel Benoit; Callet Benoit; Hedouin Valery; Gosset Didier
    Forensic science international (2003), 135 (1), 27-34 ISSN:0379-0738.
    Three modeling methods were used to estimate the time for laying of Lucilia sericata eggs (Diptera, Calliphoridae) after measurements of hatching times at several constant temperatures. These models were tested first under controlled conditions with 19 profiles of fluctuating temperatures, and on the other hand under field conditions on mice. All three models were allowed to determine the time of laying within a period of about 2h, and consequently this determines the time of death, as this species lays immediately after death under favorable conditions. Rearing of eggs sampled on a corpse may therefore contribute to the determination of a short post-mortem interval (PMI).
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3szps1GqsQ%253D%253D&md5=3aeffa41cece3bf3dafff553adeac6c4
  13. 13
    Lopez-Esclapez, R.; Garcia, M. D.; Arnaldos, M. I.; Presa, J. J.; Ubero-Pascal, N. Micron 2014, 62, 43– 51 DOI: 10.1016/j.micron.2014.03.003
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Adams, Z. J.; Hall, M. J. Forensic Sci. Int. 2003, 138, 50– 61 DOI: 10.1016/j.forsciint.2003.08.010
    Google Scholar
    14
    Methods used for the killing and preservation of blowfly larvae, and their effect on post-mortem larval length
    Adams Zoe J O; Hall Martin J R
    Forensic science international (2003), 138 (1-3), 50-61 ISSN:0379-0738.
    A record of the length of the largest larvae collected from a corpse can be used to estimate the age of the oldest larvae present and, therefore, give an estimate of minimum time since death. Consequently, factors that affect post-mortem larval length will impact on any estimate of PMI based on it. Methods used to kill and preserve larvae are known to affect post-mortem length. This study looks at the effects of different preservatives, and variations in the protocol used for killing larvae by immersion in a hot water bath = [hot water killed; HWK], on the length of dead larvae of two common blowfly species. Post-feeding third instar Calliphora vomitoria and Lucilia sericata larvae were either HWK in boiling water and then placed in 80% ethanol or 10% formaldehyde solution, or placed live into the preservatives. For both species, choice of preservative and method of killing significantly affected post-mortem length. There were significant interspecific differences in their response to identical methods of killing and preservation. Additional experiments were carried out where C. vomitoria larvae were HWK in water at 80 and 100 degrees C for 1, 30, 60 and 90 s duration. Both temperature and duration significantly affected post-mortem length. Maximum length was attained after at least 60 s immersion. The amount of post-mortem decomposition that occurred after the larvae were placed in preservative could be greatly reduced by increasing the duration of immersion and/or increasing the water temperature. For the HWK larvae, it was possible to record their length immediately after death and before they had been placed in preservative. This data revealed that where 80% ethanol was used as a preservative the larvae expanded in the preservative. The timing of this expansion was investigated with a sample of C. vomitoria, HWK at 100 degrees C for 30 s and recording post-mortem length immediately after death and again after 3, 6, 9, 12, 24, 27, 30 and 33 h storage in 80% ethanol. Maximum length was recorded after 12 h storage and the rate of expansion was highest during the first 3 h in this preservative. After long-term storage (290 days), larvae killed and preserved in the same way were on average 0.7% longer than immediately after death and 0.6% (0.11 mm) smaller than when last measured (after 28 days storage).
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3srmslChug%253D%253D&md5=729b3a51082eff57f9944a9346c1047b
  15. 15
    Day, D. M.; Wallman, J. F. Forensic Sci. Int. 2006, 159, 158– 167 DOI: 10.1016/j.forsciint.2005.07.009
    Google Scholar
    15
    Width as an alternative measurement to length for post-mortem interval estimations using Calliphora augur (Diptera: Calliphoridae) larvae
    Day Donnah M; Wallman James F
    Forensic science international (2006), 159 (2-3), 158-67 ISSN:0379-0738.
    The length of fly larvae collected from corpses is often used to help provide an entomological estimate of time since death. However, 'head-curling' by larvae can affect the accuracy of length measurements. To investigate a possible resolution to this problem, larvae of Calliphora augur were grown on sheep's liver at two constant temperatures (20 and 25 degrees C). Replicate samples were collected at set time intervals until pupation. Body length and width were measured for individual larvae and examined as predictors of age. It was found that body width, as measured at the junction of the fifth and sixth abdominal segments, is comparable with body length for age prediction of maggots of C. augur grown at these temperatures. Furthermore, conversion of width to length can be done with 95% accuracy from a simple linear model.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD283otVOguw%253D%253D&md5=5d19a30612e09d04b7e8a7d2f4bc4813
  16. 16
    Sharma, R.; Kumar Garg, R.; Gaur, J. R. Egyptian J. of Forensic Sci. 2015, 5, 1– 12 DOI: 10.1016/j.ejfs.2013.04.002
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Wells, J.; LaMotte, L. J. Forensic Sci. 1995, 40, 585– 590 DOI: 10.1520/JFS13830J
    Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Carvalho, L. M. L.; Linhares, A. X.; Trigo, J. R. Forensic Sci. Int. 2001, 120, 140– 144 DOI: 10.1016/S0379-0738(01)00421-2
    Google Scholar
    18
    Determination of drug levels and the effect of diazepam on the growth of necrophagous flies of forensic importance in southeastern Brazil
    Carvalho, L. M. L.; Linhares, A. X.; Trigo, J. R.
    Forensic Science International (2001), 120 (1-2), 140-144CODEN: FSINDR; ISSN:0379-0738. (Elsevier Science Ireland Ltd.)
    Larvae of Chrysomya albiceps (Wiedemann) and Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) were reared on tissues from rabbits administered twice the lethal dosage of diazepam in order to study the effects of this drug on the development of these two species. The rabbits were given 50 mg of diazepam via ear vein infusion. From 18 to 54 h, larvae feeding on tissues contg. the drug developed more rapidly than larvae from the control colony for both fly species. The time required for pupariation and adult emergence was significantly greater for colony fed on tissues from diazepam dosed rabbits than for the control ones. These differences are significant for they are large enough to alter the est. of postmortem interval based on fly development. The presence of diazepam could be detected through gas chromatog.-mass spectrometer (GC-MS) in all rabbit samples and in almost all diptera samples in this expt.
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  19. 19
    de Carvalho, L. M. L.; Linhares, A. X.; Badan Palhares, F. A. Forensic Sci. Int. 2012, 220, 27– 32 DOI: 10.1016/j.forsciint.2012.01.023
    Google Scholar
    19
    The effect of cocaine on the development rate of immatures and adults of Chrysomya albiceps and Chrysomya putoria (Diptera: Calliphoridae) and its importance to postmortem interval estimate
    de Carvalho Lucila Maria Lopes; Linhares Aricio Xavier; Badan Palhares Fortunato Antonio
    Forensic science international (2012), 220 (1-3), 27-32 ISSN:.
    This study aimed to determine the effect of cocaine on the development and growth of immature and adult blowflies, in an attempt to better understand the impacts of such effects on postmortem interval (PMI) estimation. Twice the lethal dose of cocaine was injected into rabbits. The control animals were injected only with saline solution. Experimental and control rabbits were autopsied, and portions of their livers were exposed to newly eclosed larvae of Chrysomya putoria and Chrysomya albiceps. Larvae were weighed individually every 6 h, up 54 h of exposure. The larvae were then placed on an artificial diet to continue their development. Pupariation time, adult emergence and adult longevity were also analysed. The larvae of both species that fed on the cocaine-containing livers developed faster than those that fed on the livers of the control animals, leading to the conclusion that cocaine influences and stimulates larval growth. The difference in growth between the control and treated flies was best observed from 12 h of exposure onward. This finding has important implications for forensic investigations.
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  20. 20
    Mai, M.; Amendt, J. Forensic Sci. Int. 2012, 221, 65– 69 DOI: 10.1016/j.forsciint.2012.04.001
    Google Scholar
    20
    Effect of different post-feeding intervals on the total time of development of the blowfly Lucilia sericata (Diptera: Calliphoridae)
    Mai Madeleine; Amendt Jens
    Forensic science international (2012), 221 (1-3), 65-9 ISSN:.
    By estimating the age of the immature stages of flies developing on a corpse, forensic entomologists are able to establish the minimum post-mortem interval. Blowflies, which are the first and most important colonizers, usually leave the cadaver at the end of the last larval stage searching for a pupation site. This period of development is referred as the post-feeding or wandering stage. The characteristics of the ground where the corpse was placed might be of notable importance for the post-feeding dispersal time: For pupariation the larvae prefer an environment protected from light and predators and may have a longer dispersal time in order to reach an appropriate pupation site. Hence, the dispersal time can vary and may influence the total time of development which may lead to an erroneous calculation of the post-mortem interval. This study investigates the effect of various post-feeding time intervals on the development of the blowfly Lucilia sericata at a temperature of 25°C. As larvae reached the post-feeding stage a pupariation substrate was offered at 0 and after 12, 24 and 48h. Only the larvae with a dispersal time of 24h (total time of development 325.2h; median) and 48h (total time of development 347.7h; median) showed a significantly longer total development time compared to the control group (total time of development 318.4h; median). The mortality rate did not differ between groups; however the flies that emerged from the group with a dispersal of 48h were significantly smaller indicating increased energy consumption during dispersal. The results of this study indicate that a prolonged post-feeding stage could increase the total developmental time of L. sericata which should be taken into consideration when interpreting entomological findings. The need for a serious examination of current rearing practices in forensic entomology laboratories is indicated because reference data sets for the time of development are usually produced by offering the post-feeding stage a substrate for pupariation immediately.
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  21. 21
    Sukontason, K.; Sukontason, K. L.; Piangjai, S.; Boonchu, N.; Kurahashi, H.; Hope, M.; Olson, J. K. Micron 2004, 35, 391– 395 DOI: 10.1016/j.micron.2003.12.004
    Google Scholar
    There is no corresponding record for this reference.
  22. 22
    Sukontason, K.; Sukontason, K. L.; Piangjai, S. Rev. Inst. Med. Trop. Sao Paulo 2003, 45, 95– 98 DOI: 10.1590/S0036-46652003000200008
    Google Scholar
    22
    Scanning electron microscopy of third-instar sarcophagid (Diptera: Sarcophagidae) recovered from a mummified human corpse in Thailand
    Sukontason Kom; Sukontason Kabkaew L; Piangjai Somsak
    Revista do Instituto de Medicina Tropical de Sao Paulo (2003), 45 (2), 95-8 ISSN:0036-4665.
    The third-instar of an unidentified sarcophagid, recovered from a mummified body of a 32-yr-old Thai male was examined using scanning electron microscopy (SEM). Although the morphological features of this larva are similar to the other sarcophagid larvae, some features could be helpful for species identification, which is a basic requirement for estimation of postmortem interval in forensic investigation. These features included number and arrangement of papillae on the anterior spiracle, structure of spines, size of circumspiracular tubercles at caudal segment and branching peculiarity of the posterior spiracular hairs. This information could benefit future identification of the sarcophagid larvae that exist in Thailand.
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  23. 23
    Whitworth, T. L.; Dawson, R. D.; Magalon, H.; Baudry, E. Proc. R. Soc. London, Ser. B 2007, 274, 1731– 1739 DOI: 10.1098/rspb.2007.0062
    Google Scholar
    23
    DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae)
    Whitworth, T. L.; Dawson, R. D.; Magalon, H.; Baudry, E.
    Proceedings of the Royal Society B: Biological Sciences (2007), 274 (1619), 1731-1739CODEN: PRSBC7 ISSN:. (Royal Society)
    In DNA barcoding, a short standardized DNA sequence is used to assign unknown individuals to species and aid in the discovery of new species. A fragment of the mitochondrial (mt) gene cytochrome c oxidase subunit 1 is emerging as the std. barcode region for animals. However, patterns of mitochondrial variability can be confounded by the spread of maternally transmitted bacteria that cosegregate with mitochondria. Here, we investigated the performance of barcoding in a sample comprising 12 species of the blow fly genus Protocalliphora, known to be infected with the endosymbiotic bacteria Wolbachia. The barcoding approach showed very limited success: assignment of unknown individuals to species is impossible for 60% of the species, while using the technique to identify new species would underestimate the species no. in the genus by 75%. This very low success of the barcoding approach is due to the non-monophyly of many of the species at the mitochondrial level. We even obsd. individuals from 4 different species with identical barcodes, which is, to our knowledge, the most extensive case of mtDNA haplotype sharing yet described. The pattern of Wolbachia infection strongly suggests that the lack of within-species monophyly results from introgressive hybridization assocd. with Wolbachia infection. Given that Wolbachia is known to infect 15-75% of insect species, we conclude that identification at the species level based on mitochondrial sequence might not be possible for many insects. However, given that Wolbachia-assocd. mtDNA introgression is probably limited to very closely related species, identification at the genus level should remain possible.
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  24. 24
    Chen, W. Y.; Hung, T. H.; Shiao, S. F. J. Med. Entomol. 2004, 41, 47– 57 DOI: 10.1603/0022-2585-41.1.47
    Google Scholar
    24
    Molecular identification of forensically important blow fly species (Diptera: Calliphoridae) in Taiwan
    Chen, Wei-Yun; Hung, Ting-Hsuan; Shiao, Shiuh-Feng
    Journal of Medical Entomology (2004), 41 (1), 47-57CODEN: JMENA6; ISSN:0022-2585. (Entomological Society of America)
    Because identification allows the proper developmental data and distribution ranges to be applied in criminal investigations, species in Taiwan were surveyed from early 2000 and were identified using mol. data. Currently, 8 species have been identified: Chrysomya megacephala, Chrysomya pinguis, Chrysomya rufifacies, Hemipyrellia ligurriens, Lucilia bazini, Lucilia cuprina, Lucilia hainanensis, and Lucilia porphyrina. We focused on classifying these blow fly species to establish a knowledge basis for further forensic entomol. research in Taiwan. Because mol. data are helpful in identifying insect specimens, esp. when no specimen of suitable condition for morphol. identification is obtained, we extd. mitochondrial cytochrome oxidase subunit I (COI) DNA of the preceding blow fly species to study its application value for their differentiation. The cloning and sequencing of the COI gene (≈1588 base pairs) of these 8 species were completed, and the data were analyzed. Preliminary results revealed the high support of congeneric groupings of species by using COI data; these sequences were also shown to be highly conserved within the same species. To actually use the database of COI sequences under various specimen conditions, specific primers were also applied for different insect stages, different segments of adults, and specimens preserved for various times. A mol. primer key was ultimately constructed for the purpose of rapid and accurate species identification at the mol. level regardless of which stage or which part of a blow fly specimen is collected.
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  25. 25
    Chen, M. S.; Wheeler, S.; Davis, H.; Whitworth, R. J.; Knutson, A.; Giles, K. L.; Royer, T. A.; Skinner, M. J. Econ. Entomol. 2014, 107, 1110– 1117 DOI: 10.1603/EC13384
    Google Scholar
    25
    Molecular markers for species identification of Hessian fly males caught on sticky pheromone traps
    Chen, Ming-Shun; Wheeler, Shanda; Davis, Holly; Whitworth, R. Jeff; Knutson, Allen; Giles, Kristopher L.; Royer, Tom A.; Skinner, Margaret
    Journal of Economic Entomology (2014), 107 (3), 1110-1117CODEN: JEENAI; ISSN:0022-0493. (Entomological Society of America)
    Pheromone traps have been widely used to monitor insect population activity. However, sticky pheromone traps for the Hessian fly (Mayetiola destructor), one of the most destructive pests of wheat, have been used only in recent years. Hessian fly male adults are small and fragile, and preserving specimens during sorting of sticky pheromone traps is a challenge when intact specimens are often required to visually distinguish them from related insects such as fungus gnats. In this study, we have established a quick and reliable method based on polymerase chain reaction markers to correctly distinguish Hessian fly males from other closely related insects. Two Hessian fly-specific markers were established, one based on the trypsin gene MDP-10 and the other based on a gene encoding the salivary gland protein SSGP31-5. Both markers provided >98% identification success of 110 Hessian fly samples prepd. from single insects. The method should provide a useful tool to allow for identification of Hessian fly individuals on sticky pheromone traps or in other situations when Hessian fly eggs, larvae, pupae, and adults are difficult to distinguish from other insects.
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  26. 26
    Wells, J. D.; Sperling, F. A. Forensic Sci. Int. 2001, 120, 110– 115 DOI: 10.1016/S0379-0738(01)00414-5
    Google Scholar
    26
    DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae)
    Wells, J. D.; Sperling, F. A. H.
    Forensic Science International (2001), 120 (1-2), 110-115CODEN: FSINDR; ISSN:0379-0738. (Elsevier Science Ireland Ltd.)
    Identifying an insect specimen is an important first step in a forensic-entomol. anal. However, diagnostic morphol. criteria are lacking for many species and life stages. We demonstrate a method for using mitochondrial DNA sequence data and phylogenetic anal. to identify any specimen of the blow fly subfamily Chrysomyinae likely to be collected from a human corpse within Canada or the USA. The reliability of the method was illustrated by analyzing specimens designed to mimic the information likely to be obtained from highly degraded specimens as well as specimens collected from widely sepd. geog. locations. Our sequence database may be suitable for another country provided the investigator knows the local fly fauna well enough to narrow the choice of chrysomyine species to those used in this study.
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  27. 27
    Avtzis, D. N.; Doudoumis, V.; Bourtzis, K. PLoS One 2014, 9, e112795 DOI: 10.1371/journal.pone.0112795
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Zhu, G. H.; Xu, X. H.; Yu, X. J.; Zhang, Y.; Wang, J. F. Forensic Sci. Int. 2007, 169, 1– 5 DOI: 10.1016/j.forsciint.2006.06.078
    Google Scholar
    28
    Puparial case hydrocarbons of Chrysomya megacephala as an indicator of the postmortem interval
    Zhu, Guang H.; Xu, Xiao H.; Yu, Xiao J.; Zhang, Yuan; Wang, Jiang F.
    Forensic Science International (2007), 169 (1), 1-5CODEN: FSINDR; ISSN:0379-0738. (Elsevier Ltd.)
    The puparial case is one of the most common stages of necrophagous flies encountered in crime investigations of highly decompd. corpses. If methods for detg. the weathering time of these puparial cases are developed, it is possible that the postmortem interval (PMI) could be estd. accordingly. Gas chromatog. coupled with mass spectrometry (GC-MS) was used to det. the changes with the weathering time in cuticular hydrocarbons of the puparial cases of C. megacephala in the lab. The results have shown that cuticular hydrocarbons of the puparial cases were a mixt. of n-alkanes, methyl-branched alkanes, and dimethyl-branched alkanes. The carbon chain length ranged from C21 to C35, and the hydrocarbon compn. showed significant regular changes with the weathering time. For the even numbered n-alkanes with low mol. wt., namely n-C22, n-C24, and n-C26, the abundance increased significantly with the weathering time. For n-C26, in particular, a linear increase in abundance with the weathering time was obsd. In addn., for most of the other low-mol.-wt. hydrocarbons (n-C26 or below), the abundance decreased significantly with the weathering time. Thus, cuticular hydrocarbon is a potential indicator of the weathering time in C. megacephala, and possibly in other necrophagous flies, and might further be used to det. the PMI.
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  29. 29
    Lavine, B. K.; Vora, M. N. J. Chromatogr A 2005, 1096, 69– 75 DOI: 10.1016/j.chroma.2005.06.049
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Ye, G.; Li, K.; Zhu, J.; Zhu, G.; Hu, C. J. Med. Entomol. 2007, 44, 450– 456 DOI: 10.1093/jmedent/44.3.450
    Google Scholar
    30
    Cuticular hydrocarbon composition in pupal exuviae for taxonomic differentiation of six necrophagous flies
    Ye, Gongyin; Li, Kai; Zhu, Jiaying; Zhu, Guanghui; Hu, Cui
    Journal of Medical Entomology (2007), 44 (3), 450-456CODEN: JMENA6; ISSN:0022-2585. (Entomological Society of America)
    Gas chromatog.-mass spectrometry was used to analyze the cuticular hydrocarbons extd. from the pupal exuviae of six necrophagous flies: Aldrichina grahami, Chrysomya megacephala, Lucilia sericata, Achoetandrus rufifacies, Boettcherisca peregrina, and Parasarcophaga crassipalpis. A discriminant model including the variables of peak 1 (tricosane), peak 7 (9-,11-,13-methyl-pentacosane), peak 21 (11,12-;9,13-dimethyl-hexacosane), peak 24 (octocosane), peak 41 (7,11-dimethyl-nonacosane), peak 42 (3-methyl-nonacosane), peak 46 (2-methyl-hentriacontane), and peak 51 (unknown) was constructed, which allowed a complete sepn. of the pupal exuviae of the six species. These results indicate that cuticular hydrocarbons as chemotaxonomic characters for insects of forensic importance are of high value and feasibility.
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  31. 31
    Page, M.; Nelson, L. J.; Blomquist, G. J.; Seybold, S. J. J. Chem. Ecol. 1997, 23, 1053– 1099 DOI: 10.1023/B:JOEC.0000006388.92425.ec
    Google Scholar
    31
    Cuticular hydrocarbons as chemotaxonomic characters of pine engraver beetles (Ips spp.) in the grandicollis subgeneric group
    Page, Marion; Nelson, Lori J.; Blomquist, Gary J.; Seybold, Steven J.
    Journal of Chemical Ecology (1997), 23 (4), 1053-1099CODEN: JCECD8; ISSN:0098-0331. (Plenum)
    Cuticular hydrocarbons were extd., identified, and evaluated as chemotaxonomic characters form all species of adult Ips pine engraver beetles in the grandicollis subgeneric group. The grandicollis group consists of Ips grandicollis (Eichhoff), I. paraconfusus Lanier, I. confusus (Le Conte), and I. hoppingi Lanier. To provide outgroups for a phylogenetic anal., cuticular hydrocarbons were also analyzed from Orthotomicus caelatus (Eichhoff), I latidens (Le Conte) (latidens subgeneric group), and I. pini (Say) (pini subgeneric group). Two hundred forty-eight hydrocarbon components were identified by gas chromatog.-mass spectrometry. The members of the grandicollis group provided 206 of these compds. The components represented eight classes: n-alkanes, alkenes, alkadienes, terminally branched methylalkanes, internally branched methylalkanes, dimethylalkanes, trimethylalkanes, and tetramethylalkanes. Different populations of O. caelatus, I. grandicollis, I. lecontei, I. montanus, I. paraconfusus, I. confusus, and I hoppingi provided no evidence for interpopulational variation in cuticular hydrocarbons. Single populations only were analyzed for I. latidens, I. pini, and I cribocollis. Sexual dimorphism in cuticular hydrocarbons occurred only in I. lecontei where females produced eight unique components with a pentatriacontane parent chain. Several phylogenetic anal. based on hydrocarbon phenotypes agreed in general with the established morphol. based system of relatedness and with published phylogenies reconstructed from protein and nucleic acid characters. Nearly all hydrocarbon anal. suggested a close relation between I. grandicollis and I. cribricollis; between I. lecontei nd I. montanus; and among the sibling species I. paraconfusus, I. lecontei and I. montanus; and among the sibling species I. paraconfusus, I confusus, and I. hoppingi. The presence or absence of specific n-alkanes (n-docosane, N-triacontane); certain dimethylalkanes (terminally branched with octacosane and triacontane parent chains and internally branched with heptacosane, hentriacontane, and docotriacontane parent chains); and 3,7,11-; 3,7,15-trimethylheptacosane permit facile discrimination of I. paraconfusus, I. confusus, and I. hoppingi. These three sibling species are difficult to resolve by external morphol. These data support the species status of I. hoppingi rather than it being considered a host race of the I. confusus complex. They also support the species status of I. cribricollis rather than it being considered part of I. grandicollis. In contrast to other published phylogenies reconstructed from mol. data, phylogenies reconstructed from cuticular hydrocarbons repeatedly place I. lecontei as an integral part of the grandicollis subgeneric group. Thus, cuticular hydrocarbon and pheromone alc. compn. of I. lecontei support its inclusion in the grandicollis subgeneric group.
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  32. 32
    Musah, R. A.; Espinoza, E. O.; Cody, R. B.; Lesiak, A. D.; Christensen, E. D.; Moore, H. E.; Maleknia, S.; Drijfhout, F. P. Sci. Rep. 2015, 5, 11520 DOI: 10.1038/srep11520
    Google Scholar
    32
    A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures
    Musah Rabi A; Lesiak Ashton D; Espinoza Edgard O; Cody Robert B; Christensen Earl D; Moore Hannah E; Drijfhout Falko P; Maleknia Simin
    Scientific reports (2015), 5 (), 11520 ISSN:.
    A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.
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  33. 33
    Rosati, J. Y.Spatial and Temporal Variability in the Carrion Insect Community: Using Blow Flies (Family: Calliphoridae) as a Model System to Study Coexistence Mechanisms at Multiple Scales. University of Windsor, Electronic Theses and Dissertations, 2014.
    Google Scholar
    There is no corresponding record for this reference.
  34. 34
    Lesiak, A. D.; Cody, R. B.; Dane, A. J.; Musah, R. A. Anal. Chem. 2015, 87, 8748– 8757 DOI: 10.1021/acs.analchem.5b01611
    Google Scholar
    There is no corresponding record for this reference.

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  2. Juan Guo, Maomao Zhang, Jian’an Liu, Rupeng Luo, Tingting Yan, Tao Yang, Xiaomei Jiang, Mengyu Dong, Yafang Yin. Evaluation of the Deterioration State of Archaeological Wooden Artifacts: A Nondestructive Protocol based on Direct Analysis in Real Time – Mass Spectrometry (DART-MS) Coupled to Chemometrics. Analytical Chemistry 2020, 92 (14) , 9908-9915. https://doi.org/10.1021/acs.analchem.0c01429
  3. Samira Beyramysoltan, Mónica I. Ventura, Jennifer Y. Rosati, Justine E. Giffen-Lemieux, Rabi A. Musah. Identification of the Species Constituents of Maggot Populations Feeding on Decomposing Remains—Facilitation of the Determination of Post Mortem Interval and Time Since Tissue Infestation through Application of Machine Learning and Direct Analysis in Real Time-Mass Spectrometry. Analytical Chemistry 2020, 92 (7) , 5439-5446. https://doi.org/10.1021/acs.analchem.0c00199
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  5. Samira Beyramysoltan, Justine E. Giffen, Jennifer Y. Rosati, Rabi A. Musah. Direct Analysis in Real Time-Mass Spectrometry and Kohonen Artificial Neural Networks for Species Identification of Larva, Pupa and Adult Life Stages of Carrion Insects. Analytical Chemistry 2018, 90 (15) , 9206-9217. https://doi.org/10.1021/acs.analchem.8b01704
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  8. Alessandra Tata, Andrea Massaro, Filippo Marzoli, Brunella Miano, Marco Bragolusi, Roberto Piro, Simone Belluco. Authentication of Edible Insects’ Powders by the Combination of DART-HRMS Signatures: The First Application of Ambient Mass Spectrometry to Screening of Novel Food. Foods 2022, 11 (15) , 2264. https://doi.org/10.3390/foods11152264
  9. Muhammad Z. Ahmed, Lily Deeter. Rapid species‐level hemolymph color test for all life stages of Nipaecoccus viridis (Newstead) (Hemiptera: Pseudococcidae), an invasive and regulatory pest in the United States. Journal of Applied Entomology 2022, 146 (4) , 454-460. https://doi.org/10.1111/jen.12985
  10. Hannah E. Moore, Martin J. R. Hall, Falko P. Drijfhout, Robert B. Cody, Daniel Whitmore. Cuticular hydrocarbons for identifying Sarcophagidae (Diptera). Scientific Reports 2021, 11 (1) https://doi.org/10.1038/s41598-021-87221-y
  11. Qiao Liu, Qirui Bi, Ninghua Tan. Authentication of three main commercial Pheretima based on amino acids analysis. Amino Acids 2021, 53 (11) , 1729-1738. https://doi.org/10.1007/s00726-021-03043-2
  12. Lauren M. Weidner, Krystal R. Hans. A review of Forensic Entomology literature in the Northeastern United States. WIREs Forensic Science 2021, 3 (5) https://doi.org/10.1002/wfs2.1402
  13. Robert B. Cody. Saccharomyces cerevisiae and S. pastorianus species and strain differentiation by direct analysis in real time time‐of‐flight mass spectrometry. Rapid Communications in Mass Spectrometry 2020, 34 (17) https://doi.org/10.1002/rcm.8835
  14. Michele C. de Paula, Kamylla B. Michelutti, Aylson D.M.M. Eulalio, Angélica Mendonça, Claudia A.L. Cardoso, Luis H.C. Andrade, Sandro M. Lima, William F. Antonialli-Junior. New approach to application of mid-infrared photoacoustic spectroscopy in forensic analysis: Study with the necrophagous blow fly Chrysomya megacephala (Diptera: Calliphoridae). Journal of Photochemistry and Photobiology B: Biology 2020, 209 , 111934. https://doi.org/10.1016/j.jphotobiol.2020.111934
  15. Justine E Giffen-Lemieux, Koji Okuda, Jennifer Y Rosati, Rabi A Musah, . Characterization of the Volatiles’ Profiles of the Eggs of Forensically Relevant Lucilia sericata and Phormia regina (Diptera: Calliphoridae) Blow Flies by SPME-Facilitated GC-MS. Journal of Medical Entomology 2020, 57 (4) , 994-1005. https://doi.org/10.1093/jme/tjaa018
  16. Éder dos Santos Souza, Richard Perosa Fernandes, Wesley Nascimento Guedes, Fábio Neves dos Santos, Marcos Nogueira Eberlin, Norberto Peporine Lopes, Victor Damasceno Padovani, João Aristeu da Rosa. Rhodnius spp. are differentiated based on the peptide/protein profile by matrix-assisted laser desorption/ionization mass spectrometry and chemometric tools. Analytical and Bioanalytical Chemistry 2020, 412 (6) , 1431-1439. https://doi.org/10.1007/s00216-019-02376-y
  17. Vandana Vinayak, Anshuman Rai. MALDITOF the Fourth Generation Techniques Still at Its Infancy to Identify Forensically Important Insects. 2020, 519-545. https://doi.org/10.1007/978-981-15-6655-4_26
  18. Joanne Y. Yew. Natural Product Discovery by Direct Analysis in Real Time Mass Spectrometry. Mass Spectrometry 2019, 8 (2) , S0081-S0081. https://doi.org/10.5702/massspectrometry.S0081
  19. Gang Lei, Fei Liu, Peng Liu, Yuan Zhou, Tong Jiao, Yong-Hui Dang. A bibliometric analysis of forensic entomology trends and perspectives worldwide over the last two decades (1998–2017). Forensic Science International 2019, 295 , 72-82. https://doi.org/10.1016/j.forsciint.2018.12.002
  20. Qing Jiang, Diya Dai, Hongli Li, David D.Y. Chen. Simultaneous determination of multiple components in cigarettes by mechanochemical extraction and direct analysis in real time mass spectrometry in minutes. Analytica Chimica Acta 2019, 62 https://doi.org/10.1016/j.aca.2019.01.024
  21. Mayara P. V. Matos, Kateryna I. Konstantynova, Rachel M. Mohr, Glen P. Jackson. Analysis of the 13C isotope ratios of amino acids in the larvae, pupae and adult stages of Calliphora vicina blow flies and their carrion food sources. Analytical and Bioanalytical Chemistry 2018, 410 (30) , 7943-7954. https://doi.org/10.1007/s00216-018-1416-9
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  • Abstract

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    Figure 1

    Figure 1. Images of eggs derived from four blow fly species from the Calliphoridae family, with the inset in each case showing a picture of the adult fly species corresponding to the eggs displayed in each panel. (A) Lucilia sericata eggs and adult fly; (B) Phormia regina eggs and adult fly; (C) L. coeruleiviridis eggs and adult fly; (D) Calliphora vicina eggs and adult fly. The scale provided refers to egg images only and does not reflect adult size.

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    Figure 2

    Figure 2. Results of DART-HRMS analyses of aqueous ethanol suspensions of six species of fly eggs: (a) C. vicina, (b) L. coeruleiviridis, (c) L. sericata, (d) P. regina, (e) Phoridae spp., and (f) Sarcophagidae spp. All analyses were performed in positive ion mode at 350 °C. Each spectrum represents an average of three analyses. The spectra generated for each species are unique and can be used as fingerprints for the identification of individual species.

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    Figure 3

    Figure 3. Head-to-tail plot showing the results of DART-HRMS in-source collision-induced dissociation experiments performed to confirm the presence of proline in P. regina eggs. The top spectrum is of the 60 V experiment of the egg sample, and the bottom spectrum is that of the amino acid analyzed at the same voltage. The presence in the egg spectrum of the peaks that appear in the spectrum of the standard indicate that the proline is present in the blow fly eggs. Both the top and bottom spectra represent an average of three analyses.

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    Figure 4

    Figure 4. Two orientations of the results of the linear discriminant analysis (LDA) plot derived from a subset of the m/z values observed in the mass spectra of ethanol suspensions of fly eggs representing the six species indicated. The data set included 90 mass spectral replicates, and 15 feature masses were used (listed in Table 2). These included some that were common to all species and others that were unique to individual species. Three principal components accounted for 93% of the variance, and the leave-one-out cross-validation (LOOCV) was 84%. The plot shows tight clustering of the data points representing samples from the same species, and clear separation between those of different species.

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  • References

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    This article references 34 other publications.

    1. 1
      Tabor, K. L.; Brewster, C. C.; Fell, R. D. J. Med. Entomol. 2004, 41, 785– 795 DOI: 10.1603/0022-2585-41.4.785
      1
      Analysis of the successional patterns of insects on carrion in southwest Virginia
      Tabor Kimberly L; Brewster Carlyle C; Fell Richard D
      Journal of medical entomology (2004), 41 (4), 785-95 ISSN:0022-2585.
      Studies of carrion-insect succession on domestic pig, Sus scrofa L., were conducted in the spring and summer of 2001 and 2002 in Blacksburg, VA, to identify and analyze the successional patterns of the taxa of forensic importance in southwest Virginia. Forty-seven insect taxa were collected in the spring. These were represented by 11 families (Diptera: Calliphoridae, Sarcophagidae, Muscidae, Sepsidae, Piophilidae; Coleoptera: Staphylinidae, Silphidae, Cleridae, Trogidae, Dermestidae, Histeridae). In the summer, 33 taxa were collected that were represented by all of the families collected in the spring, except Trogidae. The most common flies collected were the calliphorids: Phormia regina (Meigen) and Phaenicia coeruleiviridis (Macquart). The most common beetles were Creophilus maxillosus L. (Staphylinidae), Oiceoptoma noveboracense Forster, Necrophila americana L., Necrodes surinamensis (F.) (Silphidae), Euspilotus assimilis (Paykull), and Hister abbreviatus F. (Histeridae). Occurrence matrices were constructed for the successional patterns of insect taxa during 21 sampling intervals in the spring and 8 intervals in the summer studies. Jackknife estimates (mean+/-95% confidence limits) of overall Jaccard similarity in insect taxa among sampling intervals in the occurrence matrices were 0.213+/-0.081 (spring 2001), 0.194+/-0.043 (summer 2001), 0.257+/-0.068 (spring 2002), and 0.274+/-0.172 (summer 2002). Permutation analyses of the occurrence matrices showed that the patterns of succession of insect taxa were similar between spring 2001 and 2002 (P = 0.001) and between summer 2001 and 2002 (P = 0.007). The successional patterns seem to be typical for the seasonal periods and provide data on baseline fauna for estimating postmortem interval in cases of human death. This study is the first of its kind for southwest Virginia.
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    2. 2
      Tabor, K. L.; Fell, R. D.; Brewster, C. C. Forensic Sci. Int. 2005, 150, 73– 80 DOI: 10.1016/j.forsciint.2004.06.041
      2
      Insect fauna visiting carrion in Southwest Virginia
      Tabor Kimberly L; Fell Richard D; Brewster Carlyle C
      Forensic science international (2005), 150 (1), 73-80 ISSN:0379-0738.
      Successional patterns of insect fauna on pig carcasses were studied in southwest Virginia. The objective was to identify and qualitatively assess the major taxa of forensic importance in this region. Studies were conducted in spring and summer 2001 and 2002, and fall 2002. Over 50 taxa were collected and identified. Phormia regina was the dominant fly species in the spring (>90%) and co-dominant with Phaenicia coeruleiviridis in the summer. Phaenicia sericata, Lucilia illustris, and Calliphora spp. were collected in spring and summer, but less frequently. Eleven species of Sarcophagidae also were collected with Sarcophaga utilis and Helicobia rapax the most common. In the fall, the dominant fly species were Calliphora vomitoria, L. illustris, and P. coeruleiviridis. The primary beetle species collected in spring and summer included three Staphylinidae (Creophilis maxillosus, Platydracus maculosus, and Aleochara lata) and three Silphidae (Oiceoptoma noveboracense, Necrodes surinamensis, and Necrophila americana). No beetles were collected in the fall.
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    3. 3
      Pechal, J. L.; Moore, H.; Drijfhout, F.; Benbow, M. E. Forensic Sci. Int. 2014, 245, 65– 71 DOI: 10.1016/j.forsciint.2014.10.019
      3
      Hydrocarbon profiles throughout adult Calliphoridae aging: A promising tool for forensic entomology
      Pechal, Jennifer L.; Moore, Hannah; Drijfhout, Falko; Benbow, M. Eric
      Forensic Science International (2014), 245 (), 65-71CODEN: FSINDR; ISSN:0379-0738. (Elsevier Ltd.)
      Blow flies (Diptera: Calliphoridae) are typically the first insects to arrive at human remains and carrion. Predictable succession patterns and known larval development of necrophagous insects on vertebrate remains can assist a forensic entomologist with ests. of a min. post-mortem interval (PMImin) range. However, adult blow flies are infrequently used to est. the PMImin, but rather are used for a confirmation of larval species identification. Cuticular hydrocarbons have demonstrated potential for estg. adult blow fly age, as hydrocarbons are present throughout blow fly development, from egg to adult, and are stable structures. The goal of this study was to identify hydrocarbon profiles assocd. with the adults of a North American native blow fly species, Cochliomyia macellaria (Fabricius) and a North American invasive species, Chrysomya rufifacies (Macquart). Flies were reared at a const. temp. (25 °C), a photoperiod of 14:10 (L:D) (h), and were provided water, sugar and powd. milk ad libitum. Ten adult females from each species were collected at day 1, 5, 10, 20, and 30 post-emergence. Hydrocarbon compds. were extd. and then identified using gas chromatog.-mass spectrometry (GC-MS) anal. A total of 37 and 35 compds. were detected from C. macellaria and Ch. rufifacies, resp. There were 24 and 23 n-alkene and methyl-branched alkane hydrocarbons from C. macellaria and Ch. rufifacies, resp. (10 compds. were shared between species), used for statistical anal. Non-metric multidimensional scaling anal. and permutational multivariate anal. of variance were used to analyze the hydrocarbon profiles with significant differences (P < 0.001) detected among post-emergence age cohorts for each species, and unique hydrocarbon profiles detected as each adult blow fly species aged. This work provides empirical data that serve as a foundation for future research into improving PMImin ests. made by forensic practitioners and potentially increase the use of adult insects during death investigations.
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    4. 4
      Matuszewski, S.; Bajerlein, D.; Konwerski, S.; Szpila, K. Forensic Sci. Int. 2011, 207, 150– 163 DOI: 10.1016/j.forsciint.2010.09.022
      4
      Insect succession and carrion decomposition in selected forests of Central Europe. Part 3: Succession of carrion fauna
      Matuszewski Szymon; Bajerlein Daria; Konwerski Szymon; Szpila Krzysztof
      Forensic science international (2011), 207 (1-3), 150-63 ISSN:.
      The succession of insects on pig carrion was monitored in spring, summer and autumn, in three forest types and 2 years in Western Poland (Central Europe). In most forensically useful taxa, significant differences between seasons, forests and years in time of appearance on carrion were found. The lowest values of appearance time were recorded in summer and the highest in spring. In alder forest insects appeared on carcasses significantly earlier than in pine-oak forest and hornbeam-oak forest. In summer periods of insect presence on carrion were significantly shorter than in spring and autumn. In most taxa no significant effect of forest type or year on length of the presence period was found. In all seasons assemblages of adult taxa were clearly more aggregated than assemblages of larval taxa. Sequence of insects' appearance on carcasses was very similar in different seasons, forests and years. General seasonal models of insect succession on carrion are proposed for forests of Central Europe. Data on appearance time and length of the presence period in particular seasons, forests and years are presented for forensically useful taxa. Implications for methods of PMI estimation (particularly the succession-based method) are discussed.
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    5. 5
      Matuszewski, S.; Bajerlein, D.; Konwerski, S.; Szpila, K. Forensic Sci. Int. 2010, 194, 85– 93 DOI: 10.1016/j.forsciint.2009.10.016
      5
      Insect succession and carrion decomposition in selected forests of Central Europe. Part 1: Pattern and rate of decomposition
      Matuszewski Szymon; Bajerlein Daria; Konwerski Szymon; Szpila Krzysztof
      Forensic science international (2010), 194 (1-3), 85-93 ISSN:.
      Pig carrion decomposition and insect succession were monitored in different seasons and forests of Central Europe. Pattern of decomposition as well as onset, duration and rate of decompositional processes were measured. Pattern of decomposition was the same in almost all cases with putrefaction, active and advanced decay. In the majority of carcasses active decay was driven by larvae of Calliphoridae with a clear seasonal shift in dominant taxa. However, in some spring, alder forest cases active decay was driven by larvae of Necrodes littoralis (Coleoptera: Silphidae). As a rule the mosaic decomposition was observed. In spring a significant delay in onset of all decompositional processes was found. Season significantly affected rate of active decay due to a much higher rate in summer. Decomposition in alder forest proceeded faster than in pine-oak forest and hornbeam-oak forest. Differences between the latter two forests were practically negligible. Implications for forensic entomology are discussed.
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    6. 6
      Matuszewski, S.; Bajerlein, D.; Konwerski, S.; Szpila, K. Forensic Sci. Int. 2010, 195, 42– 51 DOI: 10.1016/j.forsciint.2009.11.007
      6
      Insect succession and carrion decomposition in selected forests of Central Europe. Part 2: Composition and residency patterns of carrion fauna
      Matuszewski Szymon; Bajerlein Daria; Konwerski Szymon; Szpila Krzysztof
      Forensic science international (2010), 195 (1-3), 42-51 ISSN:.
      The insect fauna of pig carcasses was monitored in different seasons and forests of Western Poland (Central Europe). The composition of carrion fauna and selected features of residency in carrion in adults and larvae of particular taxa were analysed. A total of 131 adult and 36 larval necrophilous taxa were collected. Only 51 adult species and 24 larval taxa were minimally abundant (>or=10 specimens) at least on one carcass. As for the composition of carrion fauna, there were large differences between seasons, but no important differences between forest types. In most species of Diptera, length of the presence period of adults was between 35 and 65% of the sampling interval, while in most species of Coleoptera, it was above 60%. Only in a few species (e.g., Saprinus semistriatus, Necrodes littoralis or Creophilus maxillosus) was the presence period shorter than 35% of the sampling interval. Interestingly, in some adult Coleoptera (e.g., Necrobia violacea) very long presence periods were recorded. In most taxa, the length of the presence period of larvae was between 40 and 65% of the sampling interval. Only Calliphora vomitoria, Phormia regina, Hydrotaea dentipes, N. littoralis and C. maxillosus had shorter presence periods of larvae. As a rule, residency of adults was broken, whereas residency of larvae was unbroken. Moreover, in adults, two distinct residency patterns were observed; with breaks clumped in the final part of the presence period and with breaks evenly distributed inside the presence period. Almost in all taxa, the time of appearance showed the closest relationship to the onset of bloating. The relationship was significant, positive and strong in adults of P. regina, Fannia manicata, Hydrotaea ignava, Stearibia nigriceps, S. semistriatus, N. littoralis and C. maxillosus as well as larvae of P. regina, H. dentipes, H. ignava, S. nigriceps, N. littoralis, Oiceoptoma thoracicum, Thanatophilus sp., C. maxillosus and Philonthus sp. Interestingly, in some forensically significant taxa (e.g., adults of N. violacea or Thanatophilus rugosus), we found no significant relationship between the time of their appearance and the onset of any decompositional process. Implications for the succession-based post-mortem interval (PMI) estimation, determination of the carcass movement and the season of death are discussed.
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    7. 7
      Vasconcelos, S. D.; Soares, T. F.; Costa, D. L. Int. J. Legal Med. 2014, 128, 229– 233 DOI: 10.1007/s00414-013-0936-2
      7
      Multiple colonization of a cadaver by insects in an indoor environment: first record of Fannia trimaculata (Diptera: Fanniidae) and Peckia (Peckia) chrysostoma (Sarcophagidae) as colonizers of a human corpse
      Vasconcelos Simao Dias; Soares Thiago Ferreira; Costa Diego Leonel
      International journal of legal medicine (2014), 128 (1), 229-33 ISSN:.
      We describe here a case of multiple colonization of a male cadaver found indoors in the municipality of Jaboatao dos Guararapes, Brazil. The body was colonized by six species of Diptera: Chrysomya albiceps, Chrysomya megacephala, Chrysomya putoria (Calliphoridae), Megaselia scalaris (Phoridae), Fannia trimaculata (Fanniidae), and Peckia (Peckia) chrysostoma (Sarcophagidae). The most abundant species were C. albiceps (65.0 % of all emerged adults) and C. megacephala (18.6 %). The case illustrates the ability of six insect species to simultaneously colonize a corpse in an indoor environment and represents the first collaboration between the forensic police and entomologists in Northeastern Brazil. We provide here the first record of two species, F. trimaculata and Peckia (P.) chrysostoma colonizing a human cadaver. We also report the first case of cadaver colonization by C. putoria and M. scalaris in Northeastern Brazil. Information on the development time of two species, C. albiceps and C. megacephala, were used to discuss the estimation of the post-mortem interval. Considering that the region harbors the highest rates of homicide in Brazil, implications of these findings for the consolidation of forensic entomology in the region are discussed.
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    8. 8
      Byrd, J. H.; Castner, J. L. Forensic Entomology: The Utility of Arthropods in Legal Investigations; CRC Press: Boca Raton, FL, 2002; p 681.
      There is no corresponding record for this reference.
    9. 9
      Greenberg, J. A Natural History of the Chicago Region, 2nd ed.; University of Chicago Press: Chicago, IL, 2002.
      There is no corresponding record for this reference.
    10. 10
      Sukontason, K.; Sribanditmongkol, P.; Ngoen-klan, R.; Klong-klaew, T.; Moophayak, K.; Sukontason, K. L. Parasitol. Res. 2010, 106, 641– 646 DOI: 10.1007/s00436-009-1711-7
      10
      Differentiation between Lucilia cuprina and Hemipyrellia ligurriens (Diptera: Calliphoridae) larvae for use in forensic entomology applications
      Sukontason Kom; Sribanditmongkol Pongruk; Ngoen-klan Ratchadawan; Klong-klaew Tunwadee; Moophayak Kittikhun; Sukontason Kabkaew L
      Parasitology research (2010), 106 (3), 641-6 ISSN:.
      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.
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    11. 11
      Greenberg, B.; Szyska, M. L. Ann. Entomol. Soc. Am. 1984, 77, 488– 517 DOI: 10.1093/aesa/77.5.488
      There is no corresponding record for this reference.
    12. 12
      Bourel, B.; Callet, B.; Hedouin, V.; Gosset, D. Forensic Sci. Int. 2003, 135, 27– 34 DOI: 10.1016/S0379-0738(03)00157-9
      12
      Flies eggs: a new method for the estimation of short-term post-mortem interval?
      Bourel Benoit; Callet Benoit; Hedouin Valery; Gosset Didier
      Forensic science international (2003), 135 (1), 27-34 ISSN:0379-0738.
      Three modeling methods were used to estimate the time for laying of Lucilia sericata eggs (Diptera, Calliphoridae) after measurements of hatching times at several constant temperatures. These models were tested first under controlled conditions with 19 profiles of fluctuating temperatures, and on the other hand under field conditions on mice. All three models were allowed to determine the time of laying within a period of about 2h, and consequently this determines the time of death, as this species lays immediately after death under favorable conditions. Rearing of eggs sampled on a corpse may therefore contribute to the determination of a short post-mortem interval (PMI).
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    13. 13
      Lopez-Esclapez, R.; Garcia, M. D.; Arnaldos, M. I.; Presa, J. J.; Ubero-Pascal, N. Micron 2014, 62, 43– 51 DOI: 10.1016/j.micron.2014.03.003
      There is no corresponding record for this reference.
    14. 14
      Adams, Z. J.; Hall, M. J. Forensic Sci. Int. 2003, 138, 50– 61 DOI: 10.1016/j.forsciint.2003.08.010
      14
      Methods used for the killing and preservation of blowfly larvae, and their effect on post-mortem larval length
      Adams Zoe J O; Hall Martin J R
      Forensic science international (2003), 138 (1-3), 50-61 ISSN:0379-0738.
      A record of the length of the largest larvae collected from a corpse can be used to estimate the age of the oldest larvae present and, therefore, give an estimate of minimum time since death. Consequently, factors that affect post-mortem larval length will impact on any estimate of PMI based on it. Methods used to kill and preserve larvae are known to affect post-mortem length. This study looks at the effects of different preservatives, and variations in the protocol used for killing larvae by immersion in a hot water bath = [hot water killed; HWK], on the length of dead larvae of two common blowfly species. Post-feeding third instar Calliphora vomitoria and Lucilia sericata larvae were either HWK in boiling water and then placed in 80% ethanol or 10% formaldehyde solution, or placed live into the preservatives. For both species, choice of preservative and method of killing significantly affected post-mortem length. There were significant interspecific differences in their response to identical methods of killing and preservation. Additional experiments were carried out where C. vomitoria larvae were HWK in water at 80 and 100 degrees C for 1, 30, 60 and 90 s duration. Both temperature and duration significantly affected post-mortem length. Maximum length was attained after at least 60 s immersion. The amount of post-mortem decomposition that occurred after the larvae were placed in preservative could be greatly reduced by increasing the duration of immersion and/or increasing the water temperature. For the HWK larvae, it was possible to record their length immediately after death and before they had been placed in preservative. This data revealed that where 80% ethanol was used as a preservative the larvae expanded in the preservative. The timing of this expansion was investigated with a sample of C. vomitoria, HWK at 100 degrees C for 30 s and recording post-mortem length immediately after death and again after 3, 6, 9, 12, 24, 27, 30 and 33 h storage in 80% ethanol. Maximum length was recorded after 12 h storage and the rate of expansion was highest during the first 3 h in this preservative. After long-term storage (290 days), larvae killed and preserved in the same way were on average 0.7% longer than immediately after death and 0.6% (0.11 mm) smaller than when last measured (after 28 days storage).
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    15. 15
      Day, D. M.; Wallman, J. F. Forensic Sci. Int. 2006, 159, 158– 167 DOI: 10.1016/j.forsciint.2005.07.009
      15
      Width as an alternative measurement to length for post-mortem interval estimations using Calliphora augur (Diptera: Calliphoridae) larvae
      Day Donnah M; Wallman James F
      Forensic science international (2006), 159 (2-3), 158-67 ISSN:0379-0738.
      The length of fly larvae collected from corpses is often used to help provide an entomological estimate of time since death. However, 'head-curling' by larvae can affect the accuracy of length measurements. To investigate a possible resolution to this problem, larvae of Calliphora augur were grown on sheep's liver at two constant temperatures (20 and 25 degrees C). Replicate samples were collected at set time intervals until pupation. Body length and width were measured for individual larvae and examined as predictors of age. It was found that body width, as measured at the junction of the fifth and sixth abdominal segments, is comparable with body length for age prediction of maggots of C. augur grown at these temperatures. Furthermore, conversion of width to length can be done with 95% accuracy from a simple linear model.
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    16. 16
      Sharma, R.; Kumar Garg, R.; Gaur, J. R. Egyptian J. of Forensic Sci. 2015, 5, 1– 12 DOI: 10.1016/j.ejfs.2013.04.002
      There is no corresponding record for this reference.
    17. 17
      Wells, J.; LaMotte, L. J. Forensic Sci. 1995, 40, 585– 590 DOI: 10.1520/JFS13830J
      There is no corresponding record for this reference.
    18. 18
      Carvalho, L. M. L.; Linhares, A. X.; Trigo, J. R. Forensic Sci. Int. 2001, 120, 140– 144 DOI: 10.1016/S0379-0738(01)00421-2
      18
      Determination of drug levels and the effect of diazepam on the growth of necrophagous flies of forensic importance in southeastern Brazil
      Carvalho, L. M. L.; Linhares, A. X.; Trigo, J. R.
      Forensic Science International (2001), 120 (1-2), 140-144CODEN: FSINDR; ISSN:0379-0738. (Elsevier Science Ireland Ltd.)
      Larvae of Chrysomya albiceps (Wiedemann) and Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) were reared on tissues from rabbits administered twice the lethal dosage of diazepam in order to study the effects of this drug on the development of these two species. The rabbits were given 50 mg of diazepam via ear vein infusion. From 18 to 54 h, larvae feeding on tissues contg. the drug developed more rapidly than larvae from the control colony for both fly species. The time required for pupariation and adult emergence was significantly greater for colony fed on tissues from diazepam dosed rabbits than for the control ones. These differences are significant for they are large enough to alter the est. of postmortem interval based on fly development. The presence of diazepam could be detected through gas chromatog.-mass spectrometer (GC-MS) in all rabbit samples and in almost all diptera samples in this expt.
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    19. 19
      de Carvalho, L. M. L.; Linhares, A. X.; Badan Palhares, F. A. Forensic Sci. Int. 2012, 220, 27– 32 DOI: 10.1016/j.forsciint.2012.01.023
      19
      The effect of cocaine on the development rate of immatures and adults of Chrysomya albiceps and Chrysomya putoria (Diptera: Calliphoridae) and its importance to postmortem interval estimate
      de Carvalho Lucila Maria Lopes; Linhares Aricio Xavier; Badan Palhares Fortunato Antonio
      Forensic science international (2012), 220 (1-3), 27-32 ISSN:.
      This study aimed to determine the effect of cocaine on the development and growth of immature and adult blowflies, in an attempt to better understand the impacts of such effects on postmortem interval (PMI) estimation. Twice the lethal dose of cocaine was injected into rabbits. The control animals were injected only with saline solution. Experimental and control rabbits were autopsied, and portions of their livers were exposed to newly eclosed larvae of Chrysomya putoria and Chrysomya albiceps. Larvae were weighed individually every 6 h, up 54 h of exposure. The larvae were then placed on an artificial diet to continue their development. Pupariation time, adult emergence and adult longevity were also analysed. The larvae of both species that fed on the cocaine-containing livers developed faster than those that fed on the livers of the control animals, leading to the conclusion that cocaine influences and stimulates larval growth. The difference in growth between the control and treated flies was best observed from 12 h of exposure onward. This finding has important implications for forensic investigations.
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    20. 20
      Mai, M.; Amendt, J. Forensic Sci. Int. 2012, 221, 65– 69 DOI: 10.1016/j.forsciint.2012.04.001
      20
      Effect of different post-feeding intervals on the total time of development of the blowfly Lucilia sericata (Diptera: Calliphoridae)
      Mai Madeleine; Amendt Jens
      Forensic science international (2012), 221 (1-3), 65-9 ISSN:.
      By estimating the age of the immature stages of flies developing on a corpse, forensic entomologists are able to establish the minimum post-mortem interval. Blowflies, which are the first and most important colonizers, usually leave the cadaver at the end of the last larval stage searching for a pupation site. This period of development is referred as the post-feeding or wandering stage. The characteristics of the ground where the corpse was placed might be of notable importance for the post-feeding dispersal time: For pupariation the larvae prefer an environment protected from light and predators and may have a longer dispersal time in order to reach an appropriate pupation site. Hence, the dispersal time can vary and may influence the total time of development which may lead to an erroneous calculation of the post-mortem interval. This study investigates the effect of various post-feeding time intervals on the development of the blowfly Lucilia sericata at a temperature of 25°C. As larvae reached the post-feeding stage a pupariation substrate was offered at 0 and after 12, 24 and 48h. Only the larvae with a dispersal time of 24h (total time of development 325.2h; median) and 48h (total time of development 347.7h; median) showed a significantly longer total development time compared to the control group (total time of development 318.4h; median). The mortality rate did not differ between groups; however the flies that emerged from the group with a dispersal of 48h were significantly smaller indicating increased energy consumption during dispersal. The results of this study indicate that a prolonged post-feeding stage could increase the total developmental time of L. sericata which should be taken into consideration when interpreting entomological findings. The need for a serious examination of current rearing practices in forensic entomology laboratories is indicated because reference data sets for the time of development are usually produced by offering the post-feeding stage a substrate for pupariation immediately.
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    21. 21
      Sukontason, K.; Sukontason, K. L.; Piangjai, S.; Boonchu, N.; Kurahashi, H.; Hope, M.; Olson, J. K. Micron 2004, 35, 391– 395 DOI: 10.1016/j.micron.2003.12.004
      There is no corresponding record for this reference.
    22. 22
      Sukontason, K.; Sukontason, K. L.; Piangjai, S. Rev. Inst. Med. Trop. Sao Paulo 2003, 45, 95– 98 DOI: 10.1590/S0036-46652003000200008
      22
      Scanning electron microscopy of third-instar sarcophagid (Diptera: Sarcophagidae) recovered from a mummified human corpse in Thailand
      Sukontason Kom; Sukontason Kabkaew L; Piangjai Somsak
      Revista do Instituto de Medicina Tropical de Sao Paulo (2003), 45 (2), 95-8 ISSN:0036-4665.
      The third-instar of an unidentified sarcophagid, recovered from a mummified body of a 32-yr-old Thai male was examined using scanning electron microscopy (SEM). Although the morphological features of this larva are similar to the other sarcophagid larvae, some features could be helpful for species identification, which is a basic requirement for estimation of postmortem interval in forensic investigation. These features included number and arrangement of papillae on the anterior spiracle, structure of spines, size of circumspiracular tubercles at caudal segment and branching peculiarity of the posterior spiracular hairs. This information could benefit future identification of the sarcophagid larvae that exist in Thailand.
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    23. 23
      Whitworth, T. L.; Dawson, R. D.; Magalon, H.; Baudry, E. Proc. R. Soc. London, Ser. B 2007, 274, 1731– 1739 DOI: 10.1098/rspb.2007.0062
      23
      DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae)
      Whitworth, T. L.; Dawson, R. D.; Magalon, H.; Baudry, E.
      Proceedings of the Royal Society B: Biological Sciences (2007), 274 (1619), 1731-1739CODEN: PRSBC7 ISSN:. (Royal Society)
      In DNA barcoding, a short standardized DNA sequence is used to assign unknown individuals to species and aid in the discovery of new species. A fragment of the mitochondrial (mt) gene cytochrome c oxidase subunit 1 is emerging as the std. barcode region for animals. However, patterns of mitochondrial variability can be confounded by the spread of maternally transmitted bacteria that cosegregate with mitochondria. Here, we investigated the performance of barcoding in a sample comprising 12 species of the blow fly genus Protocalliphora, known to be infected with the endosymbiotic bacteria Wolbachia. The barcoding approach showed very limited success: assignment of unknown individuals to species is impossible for 60% of the species, while using the technique to identify new species would underestimate the species no. in the genus by 75%. This very low success of the barcoding approach is due to the non-monophyly of many of the species at the mitochondrial level. We even obsd. individuals from 4 different species with identical barcodes, which is, to our knowledge, the most extensive case of mtDNA haplotype sharing yet described. The pattern of Wolbachia infection strongly suggests that the lack of within-species monophyly results from introgressive hybridization assocd. with Wolbachia infection. Given that Wolbachia is known to infect 15-75% of insect species, we conclude that identification at the species level based on mitochondrial sequence might not be possible for many insects. However, given that Wolbachia-assocd. mtDNA introgression is probably limited to very closely related species, identification at the genus level should remain possible.
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    24. 24
      Chen, W. Y.; Hung, T. H.; Shiao, S. F. J. Med. Entomol. 2004, 41, 47– 57 DOI: 10.1603/0022-2585-41.1.47
      24
      Molecular identification of forensically important blow fly species (Diptera: Calliphoridae) in Taiwan
      Chen, Wei-Yun; Hung, Ting-Hsuan; Shiao, Shiuh-Feng
      Journal of Medical Entomology (2004), 41 (1), 47-57CODEN: JMENA6; ISSN:0022-2585. (Entomological Society of America)
      Because identification allows the proper developmental data and distribution ranges to be applied in criminal investigations, species in Taiwan were surveyed from early 2000 and were identified using mol. data. Currently, 8 species have been identified: Chrysomya megacephala, Chrysomya pinguis, Chrysomya rufifacies, Hemipyrellia ligurriens, Lucilia bazini, Lucilia cuprina, Lucilia hainanensis, and Lucilia porphyrina. We focused on classifying these blow fly species to establish a knowledge basis for further forensic entomol. research in Taiwan. Because mol. data are helpful in identifying insect specimens, esp. when no specimen of suitable condition for morphol. identification is obtained, we extd. mitochondrial cytochrome oxidase subunit I (COI) DNA of the preceding blow fly species to study its application value for their differentiation. The cloning and sequencing of the COI gene (≈1588 base pairs) of these 8 species were completed, and the data were analyzed. Preliminary results revealed the high support of congeneric groupings of species by using COI data; these sequences were also shown to be highly conserved within the same species. To actually use the database of COI sequences under various specimen conditions, specific primers were also applied for different insect stages, different segments of adults, and specimens preserved for various times. A mol. primer key was ultimately constructed for the purpose of rapid and accurate species identification at the mol. level regardless of which stage or which part of a blow fly specimen is collected.
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    25. 25
      Chen, M. S.; Wheeler, S.; Davis, H.; Whitworth, R. J.; Knutson, A.; Giles, K. L.; Royer, T. A.; Skinner, M. J. Econ. Entomol. 2014, 107, 1110– 1117 DOI: 10.1603/EC13384
      25
      Molecular markers for species identification of Hessian fly males caught on sticky pheromone traps
      Chen, Ming-Shun; Wheeler, Shanda; Davis, Holly; Whitworth, R. Jeff; Knutson, Allen; Giles, Kristopher L.; Royer, Tom A.; Skinner, Margaret
      Journal of Economic Entomology (2014), 107 (3), 1110-1117CODEN: JEENAI; ISSN:0022-0493. (Entomological Society of America)
      Pheromone traps have been widely used to monitor insect population activity. However, sticky pheromone traps for the Hessian fly (Mayetiola destructor), one of the most destructive pests of wheat, have been used only in recent years. Hessian fly male adults are small and fragile, and preserving specimens during sorting of sticky pheromone traps is a challenge when intact specimens are often required to visually distinguish them from related insects such as fungus gnats. In this study, we have established a quick and reliable method based on polymerase chain reaction markers to correctly distinguish Hessian fly males from other closely related insects. Two Hessian fly-specific markers were established, one based on the trypsin gene MDP-10 and the other based on a gene encoding the salivary gland protein SSGP31-5. Both markers provided >98% identification success of 110 Hessian fly samples prepd. from single insects. The method should provide a useful tool to allow for identification of Hessian fly individuals on sticky pheromone traps or in other situations when Hessian fly eggs, larvae, pupae, and adults are difficult to distinguish from other insects.
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    26. 26
      Wells, J. D.; Sperling, F. A. Forensic Sci. Int. 2001, 120, 110– 115 DOI: 10.1016/S0379-0738(01)00414-5
      26
      DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae)
      Wells, J. D.; Sperling, F. A. H.
      Forensic Science International (2001), 120 (1-2), 110-115CODEN: FSINDR; ISSN:0379-0738. (Elsevier Science Ireland Ltd.)
      Identifying an insect specimen is an important first step in a forensic-entomol. anal. However, diagnostic morphol. criteria are lacking for many species and life stages. We demonstrate a method for using mitochondrial DNA sequence data and phylogenetic anal. to identify any specimen of the blow fly subfamily Chrysomyinae likely to be collected from a human corpse within Canada or the USA. The reliability of the method was illustrated by analyzing specimens designed to mimic the information likely to be obtained from highly degraded specimens as well as specimens collected from widely sepd. geog. locations. Our sequence database may be suitable for another country provided the investigator knows the local fly fauna well enough to narrow the choice of chrysomyine species to those used in this study.
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    27. 27
      Avtzis, D. N.; Doudoumis, V.; Bourtzis, K. PLoS One 2014, 9, e112795 DOI: 10.1371/journal.pone.0112795
      There is no corresponding record for this reference.
    28. 28
      Zhu, G. H.; Xu, X. H.; Yu, X. J.; Zhang, Y.; Wang, J. F. Forensic Sci. Int. 2007, 169, 1– 5 DOI: 10.1016/j.forsciint.2006.06.078
      28
      Puparial case hydrocarbons of Chrysomya megacephala as an indicator of the postmortem interval
      Zhu, Guang H.; Xu, Xiao H.; Yu, Xiao J.; Zhang, Yuan; Wang, Jiang F.
      Forensic Science International (2007), 169 (1), 1-5CODEN: FSINDR; ISSN:0379-0738. (Elsevier Ltd.)
      The puparial case is one of the most common stages of necrophagous flies encountered in crime investigations of highly decompd. corpses. If methods for detg. the weathering time of these puparial cases are developed, it is possible that the postmortem interval (PMI) could be estd. accordingly. Gas chromatog. coupled with mass spectrometry (GC-MS) was used to det. the changes with the weathering time in cuticular hydrocarbons of the puparial cases of C. megacephala in the lab. The results have shown that cuticular hydrocarbons of the puparial cases were a mixt. of n-alkanes, methyl-branched alkanes, and dimethyl-branched alkanes. The carbon chain length ranged from C21 to C35, and the hydrocarbon compn. showed significant regular changes with the weathering time. For the even numbered n-alkanes with low mol. wt., namely n-C22, n-C24, and n-C26, the abundance increased significantly with the weathering time. For n-C26, in particular, a linear increase in abundance with the weathering time was obsd. In addn., for most of the other low-mol.-wt. hydrocarbons (n-C26 or below), the abundance decreased significantly with the weathering time. Thus, cuticular hydrocarbon is a potential indicator of the weathering time in C. megacephala, and possibly in other necrophagous flies, and might further be used to det. the PMI.
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    29. 29
      Lavine, B. K.; Vora, M. N. J. Chromatogr A 2005, 1096, 69– 75 DOI: 10.1016/j.chroma.2005.06.049
      There is no corresponding record for this reference.
    30. 30
      Ye, G.; Li, K.; Zhu, J.; Zhu, G.; Hu, C. J. Med. Entomol. 2007, 44, 450– 456 DOI: 10.1093/jmedent/44.3.450
      30
      Cuticular hydrocarbon composition in pupal exuviae for taxonomic differentiation of six necrophagous flies
      Ye, Gongyin; Li, Kai; Zhu, Jiaying; Zhu, Guanghui; Hu, Cui
      Journal of Medical Entomology (2007), 44 (3), 450-456CODEN: JMENA6; ISSN:0022-2585. (Entomological Society of America)
      Gas chromatog.-mass spectrometry was used to analyze the cuticular hydrocarbons extd. from the pupal exuviae of six necrophagous flies: Aldrichina grahami, Chrysomya megacephala, Lucilia sericata, Achoetandrus rufifacies, Boettcherisca peregrina, and Parasarcophaga crassipalpis. A discriminant model including the variables of peak 1 (tricosane), peak 7 (9-,11-,13-methyl-pentacosane), peak 21 (11,12-;9,13-dimethyl-hexacosane), peak 24 (octocosane), peak 41 (7,11-dimethyl-nonacosane), peak 42 (3-methyl-nonacosane), peak 46 (2-methyl-hentriacontane), and peak 51 (unknown) was constructed, which allowed a complete sepn. of the pupal exuviae of the six species. These results indicate that cuticular hydrocarbons as chemotaxonomic characters for insects of forensic importance are of high value and feasibility.
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    31. 31
      Page, M.; Nelson, L. J.; Blomquist, G. J.; Seybold, S. J. J. Chem. Ecol. 1997, 23, 1053– 1099 DOI: 10.1023/B:JOEC.0000006388.92425.ec
      31
      Cuticular hydrocarbons as chemotaxonomic characters of pine engraver beetles (Ips spp.) in the grandicollis subgeneric group
      Page, Marion; Nelson, Lori J.; Blomquist, Gary J.; Seybold, Steven J.
      Journal of Chemical Ecology (1997), 23 (4), 1053-1099CODEN: JCECD8; ISSN:0098-0331. (Plenum)
      Cuticular hydrocarbons were extd., identified, and evaluated as chemotaxonomic characters form all species of adult Ips pine engraver beetles in the grandicollis subgeneric group. The grandicollis group consists of Ips grandicollis (Eichhoff), I. paraconfusus Lanier, I. confusus (Le Conte), and I. hoppingi Lanier. To provide outgroups for a phylogenetic anal., cuticular hydrocarbons were also analyzed from Orthotomicus caelatus (Eichhoff), I latidens (Le Conte) (latidens subgeneric group), and I. pini (Say) (pini subgeneric group). Two hundred forty-eight hydrocarbon components were identified by gas chromatog.-mass spectrometry. The members of the grandicollis group provided 206 of these compds. The components represented eight classes: n-alkanes, alkenes, alkadienes, terminally branched methylalkanes, internally branched methylalkanes, dimethylalkanes, trimethylalkanes, and tetramethylalkanes. Different populations of O. caelatus, I. grandicollis, I. lecontei, I. montanus, I. paraconfusus, I. confusus, and I hoppingi provided no evidence for interpopulational variation in cuticular hydrocarbons. Single populations only were analyzed for I. latidens, I. pini, and I cribocollis. Sexual dimorphism in cuticular hydrocarbons occurred only in I. lecontei where females produced eight unique components with a pentatriacontane parent chain. Several phylogenetic anal. based on hydrocarbon phenotypes agreed in general with the established morphol. based system of relatedness and with published phylogenies reconstructed from protein and nucleic acid characters. Nearly all hydrocarbon anal. suggested a close relation between I. grandicollis and I. cribricollis; between I. lecontei nd I. montanus; and among the sibling species I. paraconfusus, I. lecontei and I. montanus; and among the sibling species I. paraconfusus, I confusus, and I. hoppingi. The presence or absence of specific n-alkanes (n-docosane, N-triacontane); certain dimethylalkanes (terminally branched with octacosane and triacontane parent chains and internally branched with heptacosane, hentriacontane, and docotriacontane parent chains); and 3,7,11-; 3,7,15-trimethylheptacosane permit facile discrimination of I. paraconfusus, I. confusus, and I. hoppingi. These three sibling species are difficult to resolve by external morphol. These data support the species status of I. hoppingi rather than it being considered a host race of the I. confusus complex. They also support the species status of I. cribricollis rather than it being considered part of I. grandicollis. In contrast to other published phylogenies reconstructed from mol. data, phylogenies reconstructed from cuticular hydrocarbons repeatedly place I. lecontei as an integral part of the grandicollis subgeneric group. Thus, cuticular hydrocarbon and pheromone alc. compn. of I. lecontei support its inclusion in the grandicollis subgeneric group.
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    32. 32
      Musah, R. A.; Espinoza, E. O.; Cody, R. B.; Lesiak, A. D.; Christensen, E. D.; Moore, H. E.; Maleknia, S.; Drijfhout, F. P. Sci. Rep. 2015, 5, 11520 DOI: 10.1038/srep11520
      32
      A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures
      Musah Rabi A; Lesiak Ashton D; Espinoza Edgard O; Cody Robert B; Christensen Earl D; Moore Hannah E; Drijfhout Falko P; Maleknia Simin
      Scientific reports (2015), 5 (), 11520 ISSN:.
      A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.
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    33. 33
      Rosati, J. Y.Spatial and Temporal Variability in the Carrion Insect Community: Using Blow Flies (Family: Calliphoridae) as a Model System to Study Coexistence Mechanisms at Multiple Scales. University of Windsor, Electronic Theses and Dissertations, 2014.
      There is no corresponding record for this reference.
    34. 34
      Lesiak, A. D.; Cody, R. B.; Dane, A. J.; Musah, R. A. Anal. Chem. 2015, 87, 8748– 8757 DOI: 10.1021/acs.analchem.5b01611
      There is no corresponding record for this reference.

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