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

López-Esclapez, Raquel; García, María de las Nieves González; Arnaldos, Ma Isabel; Presa, Juan José; Ubero-Pascal, Nicolás

doi:10.1016/j.micron.2014.03.003

Cited by 5 publications

(4 citation statements)

References 77 publications

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“…The immature specimens selected, except puparia, were rinsed and euthanized in water near to boiling according to EAFE guidelines (Amendt et al, ), and fixed in McDowell fixative solution (4% formalin, 1% glutaraldehyde mixture in cacodylate buffer solution, pH 7.4), at 4°C for 24 h. Afterward, the specimens were rinsed again twice in sodium cacodylate buffer solution 0.1 M. For SEM analysis, 40 specimens (15 larvae each of instars I and II and five specimens each of third instar larvae and puparia) were dehydrated until absolute acetone in a gradient of increasing concentration of ethanol and ethanol–acetone solution (50% of each reagent) and air‐dried after hexamethyldisilazane treatment (López‐Esclapez et al, ). Dried specimens were mounted on SEM stubs with conductive adhesive tape, mounting them in different positions (dorsal, lateral, and ventral views).…”

Section: Methodsmentioning

confidence: 99%

Micromorphology of immature stages of Sarcophaga (Liopygia) cultellataPandellé, 1896 (Diptera: Sarcophagidae), a forensically important fly

Ubero-Pascal

Paños

García

et al. 2014

Microscopy Res & Technique

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The Sarcophagidae are one of the most numerous groups of Diptera in the world, consisting of many species of forensic interest, whose immature stages are useful in the estimation of postmortem interval. The immature stages of some species of forensic importance still remain unknown or undescribed, like in the case of Sarcophaga (Liopygia) cultellata Pandellé, 1986, a species restricted to the Iberian Peninsula, south of France and north of Italy, which shares a ecological niche with species of the same subgenus, e.g., Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830) and Sarcophaga (Liopygia) crassipalpis Macquart, 1839, making it necessary to lay the groundwork for a proper specific differentiation before it can be successfully applied in forensic practice. This study provides the first micromorphological description of all preimaginal stages of S. (L.) cultellata using light microscopy and scanning electron microscopy (SEM), the results of which allow the morphology of the main features to be followed during the immature life cycle. We propose a combination of features for distinguishing Liopygia from other sarcophagid subgenera, based on the current level of morphological knowledge of immature stages. S. (L.) cultellata can be differentiated from S. (L.) argyrostoma and S. (L.) crassipalpis in every immature stage by both light microscopy and SEM. The presence of tegumental warts and a fan-shaped anterior spiracle with a single row of 15-18 respiratory papillae allow distinguishing the third instar larvae of S. (L.) cultellata from other Sarcophaga species described hitherto by SEM. Identification keys based on light microscopy observations are provided, covering all the immature stages of Liopygia subgenus occurring in the Iberian Peninsula.

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Section: Methodsmentioning

confidence: 99%

Micromorphology of immature stages of Sarcophaga (Liopygia) cultellataPandellé, 1896 (Diptera: Sarcophagidae), a forensically important fly

Ubero-Pascal

Paños

García

et al. 2014

Microscopy Res & Technique

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“…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.…”

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confidence: 99%

“…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 . Importantly, eggs retrieved from corpses are often suspended in aqueous ethanol on-site, which precludes the possibility of rearing them to adulthood. , 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. − 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. − 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.…”

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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

et al. 2017

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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.

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“…While the aforementioned approaches to the analysis of insect life stages were performed on hexane extracts, an analysis method that utilizes aqueous ethanol solutions in which the samples have been immersed would be much more efficient, since this is the form in which the samples are generated in the field and the manner in which they are stored. , The standard aqueous ethanol solution routinely used for long-term storage of insect evidence provides a medium that can easily and rapidly be analyzed by DART-HRMS to reveal unique and diagnostic chemical fingerprints, including amino acids profiles that are characteristic for a given species . In addition, although previous studies exploited the use of multivariate discriminant analysis techniques to distinguish between species, a statistical analysis method that enables differentiation and identification of a range of species in their different life stages needs to be demonstrated and developed in order for this approach to have practical utility.…”

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confidence: 99%

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

et al. 2018

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Species determination of the various life stages of flies (Order: Diptera) is challenging, particularly for the immature forms, because analogous life stages of different species are difficult to differentiate based on morphological features alone. It is demonstrated here that direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) combined with supervised Kohonen Self-Organizing Maps (SOM) enables accomplishment of species-level identification of larva, pupa, and adult life stages of carrion flies. DART-HRMS data for each life stage were acquired from analysis of ethanol suspensions representing Calliphoridae, Phoridae, and Sarcophagidae families, without additional sample preparation. After preprocessing, the data were subjected to a combination of minimum Redundancy Maximal Relevance (mRMR) and Sparse Discriminant Analysis (SDA) methods to select the most significant variables for creating accurate SOM models. The resulting data were divided into training and validation sets and then analyzed by the SOM method to define the proper discrimination models. The 5-fold venetian blind cross-validation misclassification error was below 7% for all life stages, and the validation samples were correctly identified in all cases. The multiclass SOM model also revealed which chemical components were the most significant markers for each species, with several of these being amino acids. The results show that processing of DART-HRMS data using artificial neural networks (ANNs) based on the Kohonen SOM approach enables rapid discrimination and identification of fly species even for the immature life stages. The ANNs can be continuously expanded to include a larger number of species and can be used to screen DART-HRMS data from unknowns to rapidly determine species identity.

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

Cited by 5 publications

References 77 publications

Micromorphology of immature stages of Sarcophaga (Liopygia) cultellataPandellé, 1896 (Diptera: Sarcophagidae), a forensically important fly

Micromorphology of immature stages of Sarcophaga (Liopygia) cultellataPandellé, 1896 (Diptera: Sarcophagidae), a forensically important fly

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

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

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