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EFFECT OF ALCOHOL-BASED PRESERVATIVES ON QUALITY OF BEETLE LARVAL SPECIMENS
Abstract
The standard procedure for fluid preservation of insects consists of two steps. First, the specimen is killed and prepared in a fixative fluid and then transferred to a storage fluid. Recommendations for using specific chemicals or mixtures differ depending on the size and anatomy of the animal and vary among the authors. However, controlled comparative studies are often missing. The ideal method should preserve the specimen's morphology as well as its genetic information. We have investigated the effect of 12 combinations of commonly used methods based on ethanol, using four types of fixatives (Hot Water Kill (HWK) for 5 minutes/6 hours; absolute ethanol cold/boiling) and three types of storage fluids (70 %; 80 %; and absolute ethanol) after eight months of storage. The effects were tested on mature larvae of a forensically relevant beetle species. We have evaluated the effectivity in terms of color preservation, shape of the specimen, and quality of genetic material. The color was best preserved by HWK for 5 min with subsequent storage in absolute ethanol, and the worst results were achieved by cold absolute ethanol with subsequent storage in 70 % ethanol. The fixative type has a major effect on the shape compared to the storage fluid. The effect of treatments on shape appears to be inverse to the effect on color preservation. The genetic material was preserved equally well in all treatments. The advantages and disadvantages of specific treatments and the recommendation of the best method are discussed.
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Insect pupae sampled at a death scene may be used to estimate the post-mortem interval. The pupal age is however difficult to estimate, as there are no good quantitative markers for the age of a pupa. We present a novel method for pupal age estimation based on the quantification of contrast in intensity between the eyes of a pupa and the middle grey photography card as a standard background. The intensity is measured on a standardized scale from 0 (perfect black) to 255 (perfect white) using computer graphical software and pictures of the eye and the background taken with a stereomicroscope. Eye-background contrast is calculated by subtracting the average intensity of the eye from the average intensity of the background. The method was developed and validated using pupae of Necrodes littoralis (Linnaeus, 1758) (Coleoptera: Silphidae), one of the most abundant beetle species on human cadavers in Central Europe. To develop the model, pupae were reared in 17, 20 and 23 °C, with a total of 120 specimens. The method was validated by three raters, using in total 182 pupae reared in 15, 17, 20, 23 and 25 °C. We found a gradual increase in eye-background contrast with pupal age. Changes followed generalized logistic function, with almost perfect fit of the model. Using our method pupal age was estimated with the average error of 8.1 accumulated degree-days (ADD). The largest error was 27.8 ADD and 95% of age estimates had errors smaller than 20 ADD. While using the method, different raters attained similar accuracy. In conclusion, we have demonstrated that eye-background contrast is a good quantitative marker for the age of N. littoralis pupae. Contrast measurements gave accurate estimates for pupal age. Our method is thus proven to be a candidate for a reliable approach to age insect pupae in forensic entomology.
The molecular evolutionary genetics analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.
Fluid preservation refers to specimens and objects that are preserved in fluids, most commonly alcohol and formaldehyde, but also glycerin, mineral oil, acids, glycols, and a host of other chemicals that protect the specimen from deterioration. Some of the oldest natural history specimens in the world are preserved in fluid.
Despite the fact that fluid preservation has been practiced for more than 350 years, this is the only handbook that summarize all that is known about this complex and often confusing topic. Fluid Preservation: A Comprehensive Reference covers the history and techniques of fluid preservation and how to care for fluid preserved specimens in collections.
More than 900 references on fluid preservation were reviewed for this project.
An historical survey of preservative recipes provides for guidance for museums with older collections (many fluid preservatives contain hazardous chemicals).
Current standards and best practices for collection care and management are presented.
Current and controversial topics (e.g., the preservation of DNA, alternatives to alcohol and formaldehyde) are discussed and fully referenced.
Health and safety issues involved with caring for fluid preserved collections are discussed.
The final chapter addresses fluid preserved specimens as cultural products and their use in art, literature, film, and song.
Although most fluid-preserved specimens are found in natural history and medical museums, it is not at all uncommon to find them in art museums, history museums, and science centers. In addition to animals, plants, and anatomical specimens, fluid preserved collections include some minerals and fossils and many other objects.
Fluid Preservation is an essential reference for:
Natural history curators
Natural history collections managers
Conservators
Medical and anatomical museum collections managers and curators
Art and history museum staff who have fluid preserved specimens and objects in their care (e.g., works by Damien Hirst)
Private collectors
Researchers using museum collections as sources of DNA, isotopes, etc.
Health and safety professionals
Exhibit planners and designers
Museum facilities planners and managers
People interested in the history of science
People interested in the history of natural history museums
Museum studies students
Forensic entomology, the use of insects and other arthropods in forensic investigations, is becoming increasingly more important in such investigations. To ensure its optimal use by a diverse group of professionals including pathologists, entomologists and police officers, a common frame of guidelines and standards is essential. Therefore, the European Association for Forensic Entomology has developed a protocol document for best practice in forensic entomology, which includes an overview of equipment used for collection of entomological evidence and a detailed description of the methods applied. Together with the definitions of key terms and a short introduction to the most important methods for the estimation of the minimum postmortem interval, the present paper aims to encourage a high level of competency in the field of forensic entomology.
Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. C SIMON, F FRATI, A BECKENBACH, B CRESPI, H LIU, P
Numerous papers have been published recently regarding deoxyribonucleic acid (DNA) studies of museum insect specimens. Despite the great enthusiasm for such analyses, there continues to be a potential conflict between specimen maintenance and their use for experimental procedures, since DNA extraction methods are sample destructive. This has created a strong limitation for studies on museum specimens, making it impossible to work on rare or irreplaceable species. However, new methods for improving insect preservation have been recently published, together with new procedures for performing less destructive DNA extractions that combine specimen maintenance and DNA extraction.
Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson’s II, 70% ethanol, UMFIX, modified Carnoy’s, modified methacarn, Bouin’s, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy’s solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy’s solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.
In attempts to establish a convenient and reliable method for field collection and archival preservation of insects and their endosymbiotic microorganisms for molecular analysis, acetone, ethanol, and other organic solvents were tested for DNA preservability of the pea aphid Acyrthosiphon pisum and its intracellular symbiotic bacterium Buchnera sp. After 6 months’ storage, not only the band of high-molecular-size DNA but also the bands of rRNA were well preserved in acetone, ethanol, 2-propanol, diethyl ether and ethyl acetate. Polymerase chain reaction (PCR) assays confirmed that the DNA of both the insects and their symbionts was well preserved in these solvents. In contrast, methanol and chloroform showed poor DNA preservability. When water-containing series of acetone and ethanol were examined for DNA preservability, acetone was apparently more robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be a more recommendable preservative for DNA analysis than ethanol which has been widely used for this purpose. The DNA of various insects could be preserved in acetone at room temperature in good condition for several years. In addition to the DNA of the host insects, the DNA of their endosymbionts, including Buchnera and other mycetocyte symbionts, Wolbachia, and gut bacteria, was amplified by PCR after several years of acetone storage. The RNA and protein of the pea aphid and its endosymbiont were also preserved for several years in acetone. After 2 years’ storage in acetone, proteins of A. pisum could be analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting, and the endosymbiotic bacteria were successfully detected by immunohistochemistry and in situ hybridization on the tissue sections.
A major role of forensic entomology is to estimate the post-mortem interval. An entomologist's estimate of post-mortem interval is based on a series of generally valid assumptions, error in any of which can alter the accuracy of an estimate. The initial process of collecting and preserving maggots can itself lead to error, as can the method of killing and preservation. Since circumstances exist where it is not possible to rear maggots, methods of killing and preservation can be vital to preserving the integrity of entomological evidence. In this study, a number of preservation techniques used at crime scenes and in mortuaries were examined, and their effect on feeding third-instar larvae of Calliphora augur and Lucilia cuprina evaluated. The preservatives used were 70, 75, 80, 90 and 100% EtOH, Kahle's solution and 10% formalin. Each treatment was replicated three times. The effect of handling on first- and second-instar, feeding and post-feeding third-instar larvae of C. augur was also examined and compared to unhandled controls. Finally, the effects of preservatives were noted when larvae of C. augur and L. cuprina were placed into preservatives alive. It was found that continued handling is detrimental to specimens because preservative evaporates from both the vial and the specimens. No single preservative type was found to be entirely suitable for both species if DNA retrieval is desired. Specimens placed into most preservatives alive exhibited adverse colour changes, desiccation, sunkeness and agglomeration. It is concluded that the reaction to preservative type might be species specific and that different instars of the same species might also react differently.
Entomological Evidence Collections Methods: American Board of Forensic Entomology Approved Protocols
- M R Sanford
- J H Byrd
- J K Tomberlin
- J R Wallace
Sanford M.R., Byrd J.H., Tomberlin J.K. & Wallace J.R. Entomological Evidence
Collections Methods: American Board of Forensic Entomology Approved Protocols. In
Byrd J.H. & Tomberlin J.K. (Eds). Forensic Entomology, The Utility of Arthropods in
Legal Investigations, Third Edition. CRC Press, Taylor & Francis Group, pp 63-86,
2020.
Directions for Collecting and Preserving Insects (Classic Reprint). Forgotten Books
- C V Riley
Riley C.V. Directions for Collecting and Preserving Insects (Classic Reprint).
Forgotten Books, 162 pp, 1892.