Instant Insect Identification to Aid Forensic Entomology Investigations

Instant Insect Identification to Aid Forensic Entomology Investigations

During the investigation of a suspicious death, entomological (that is, insect-related) evidence may be able to provide vital clues as to when the victim died. Determining time since death, or post-mortem interval, can be one of the most important aspects of such an investigation, so it comes as no surprise that a great deal of research has been directed towards improving these estimations.

Insects can play a huge role in estimating time since death. Various types of species of insect will often visit the scene of a death in a relatively predictive manner, either to feed on the decomposing remains (known as necrophagous insects), to prey on other insects present, or to find a suitable place to lay their eggs. Blow flies, a group which includes common flies such as the bluebottle and the greenbottle, are often of particular interest. Forensic entomologists will typically study the insects, eggs and larvae present at a death scene, utilising the type of bugs found and their stage of development to track back to the likely time at which they arrived, thus when the victim may have died. However in order to accurately do this, entomologists must often collect insect specimens for closer inspection and even to rear to adulthood in order to determine the exact species, which is evidently a time-consuming process requiring a high level of expert knowledge.

For the first time, researchers at the University of Albany have applied a technique called direct analysis in real time with high resolution mass spectrometry, or DART-HRMS for short, to the analysis of blow fly eggs. Published in the latest issue of the journal Analytical Chemistry, the technique has demonstrated the possibility of almost instantly differentiating between different fly species based on the amino acid profiles of the eggs.

DART-MS, developed in 2005 by Dr Chip Cody of JEOL, is an ambient ionisation mass spectrometry technique that allows for samples to be directly analysed without any time-consuming sample preparation steps, and perhaps most importantly without destroying the sample. The sample is simply presented in its native state between the ion source and the inlet of the mass spectrometer, enabling compounds present in the sample to be ionised and drawn into the instrument for analysis and identification.

dartms

Sampling interface of DART-MS. Source: Wikimedia Commons

During this investigation, researchers used pieces of pork liver to attract a number of different blow fly species before transporting them to the laboratory. The flies were reared until they lay new eggs, which would be the focus of the analysis. The study utilised specimens of a number of species, including Calliphora vicinia, Lucilia coeruleiviridis, Lucilia sericata, Phormia regina, along with specimens from the Phoridae and Sarcophagidae families. Even to the eye of an expert, the eggs of these specimens are often indistinguishable. The eggs were simply placed in an ethanol solution and the mixtures directly subjected to DART-HRMS analysis.

The technique focused on the analysis and identification of amino acids in the eggs, essentially enabling researchers to produce a chemical fingerprint unique to eggs of a particular species. Examination of the mass spectra showed that the different species exhibited a unique chemical fingerprint, and by using multivariate analysis it was possible to better visualise the similarities and differences between amino acids detected in the eggs of different species.

Unsurprisingly, many amino acids were common to multiple species. For instance, alanine, isoleucine and proline were detected in four of the species, whereas valine was detected in all but one of the egg samples. However some compounds were unique to particular species, and it is these unique amino acids that will prove to be most beneficial in differentiating between the eggs of different species. For instance, glutamine and tryptophan were only present in the eggs belonging to P. regina. Interestingly, the research also demonstrated the ability to distinguish between families as well as species, with some compounds only detected in the eggs of specific families.

By using this particular technique, almost instantaneous identification could be achieved. Of course this research has included only a very limited number of species, thus a much bigger investigation would be necessary before the technique would really be beneficial to a legal investigation. Not only would further species need to be included, but another potential development would be the production of a chemical profile database against which unknown insect samples could be compared. Developed further, the use of DART-MS could save investigators a lot of time in the identification of insects of forensic interest.

 

References

Cody, R. B., Laramée, J. A. & Durst, H. D. Versatile New Ion Source for the Analysis of Materials in Open Air under Ambient Conditions. Anal. Chem. 77, 2297–2302 (2005).

Giffen, J. E., Rosati, J. Y., Longo, C. M. & Musah, R. A. Species Identification of Necrophagous Insect Eggs Based on Amino Acid Profile Differences Revealed by Direct Analysis in Real Time-High Resolution Mass Spectrometry. Anal. Chem. (2017) In Press

 

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Killer Cocktails: The Chemistry Behind the Lethal Injection

Killer Cocktails: The Chemistry Behind the Lethal Injection

In many countries worldwide, including the United States, lethal injection is used as a humane method of executing a death row inmate. With the lethal injection, the life of the inmate can theoretically be cleanly and swiftly ended through administering a number of drugs, with no pain and minimal trauma.

The debate over the lethal injection hit the news again last month when the U.S. Supreme Court ruled against claims that the use of a drug used in lethal injections (midazolam hydrochloride) violates the Eighth Amendment (relating to prohibiting cruel and unusual punishment). Despite this method of capital punishment largely replacing supposedly less humane forms of death such as the electric chair and hanging, there is still great debate over the ethics of certain drugs used, and whether they actually do provide a swift and pain-free death.

But what drugs are involved in this lethal cocktail, and how do these end life in an apparently ethical manner?

The procedure for lethal injection can vary across different countries and even different states. In the United States, execution by lethal injection is typically achieved through the intravenous use of three drugs in succession, each with a different purpose, though in some instances a single-drug method is used, usually involving a lethal dose of anaesthetic.

Sodium Thiopental (Source: Chemspider)

Sodium Thiopental (Source: Chemspider)

But let’s look at the three-part cocktail. The first drug to be administered is usually a barbiturate to act as an anaesthetic (painkiller), used to ensure the remaining steps in the procedure do not cause any pain. Traditionally sodium thiopental is used, a fast-onset but short-acting barbiturate. Barbiturates are compounds which can ultimately produce anaesthetic effects. They act as agonists of gamma-aminobutyric acid (GABA) receptors, which are inhibitory neurotransmitters in the central nervous system. By binding to this receptor, the activity of the central nervous system is depressed, bringing about effects ranging from mild sedation to general anaesthesia. In this instance, a sufficient dosage is administered to render the inmate unconscious, thus ensuring a painless procedure. However some have argued that the fast-acting effects of sodium thiopental can wear off before the execution procedure is complete.

Succinylcholine Chloride (Source: Chemspider)

Succinylcholine Chloride (Source: Chemspider)

Once the inmate is unconscious, a neuromuscular-blocking drug is then administered, generally succinylcholine (also known as suxamethonium chloride) or pancuronium bromide. Compounds such as succinylcholine bind to acetylcholine receptors, blocking the action of acetylcholine, a neurotransmitter essential in the proper functioning of skeletal muscle. When succinylcholine binds to this receptor, a cation channel in the receptor opens and depolarisation of the neuromuscular junction occurs. Normally when acetylcholine binds to this receptor, it soon dissociates following depolarisation and the muscle cell will be ready for the next signal. However compounds such as succinylcholine have a significantly longer duration, ultimately resulting in paralysis. In short, administering a drug such as succinylcholine prevents acetylcholine from communicating with the muscles and thus paralyses the inmate’s muscles, including those used to breathe. Other drugs such as pancuronium bromide can also be used, which have a different mechanism of action but ultimately achieve the same final result of muscle paralysis.

Finally the salt potassium chloride is administered. Within the body a variety of salts are vital for brain function, transmission of nerve signals and the beating of the heart, and these salt levels are tightly regulated by the body. In the normal functioning of the body, the majority of potassium is confined to the cells, with very little being present in the bloodstream at any one time. The introduction of a large amount of potassium chloride disrupts this electrochemical balance as the body’s cell are not able to equilibrate, rendering the cells unable to function, leading to cardiac arrest. In simpler terms, the overdose of potassium chloride brings about a condition known as hyperkalemia, in which the potassium concentration in the body is too high, causing the heart to fail. The inmate is officially declared dead when a cardiac monitor indicates the heart has stopped.

Recently, the drug used to initially render the inmate unconscious, sodium thiopental, has been difficult to obtain for a number of reasons, thus some states in the U.S. have used midazolam hydrochloride, a drug which has ultimately caused a great deal of controversy in recent years, such as in the Clayton Lockett case. This benzodiazepine is commonly used as a sedative, but when used during the lethal injection procedure, it is generally combined with an opiate. This is because midazolam itself has no analgesic (painkilling) effect, thus an additional drug is required to achieve this. Despite its recent use, claims have been made that a number of executions using this drug resulted in the prisoners showing signs of consciousness and gasping, suggesting that they were not quite as unconscious as intended. If the inmate is not unconscious when the muscle paralyser and electrolytes are administered, they may experience suffocation due to the muscle paralysing agent and burning caused by the potassium chloride.

So there we have it – some of the primary drugs administered during the lethal injection procedure and how they react within the body to bring about death. For more information on the death penalty (namely in the U.S), visit the Death Penalty Information Center.

References

Johnson, B. A. 2011. Addiction Medicine: Science and Practice Volume 1. New York: Springer.

Kroll, D. 2014. The Drugs Used in Execution by Lethal Injection. [online] Available from: http://www.forbes.com/sites/davidkroll/2014/05/01/the-pharmacology-and-toxicology-of-execution-by-lethal-injection

Kemsley, J. 2015. Sedative for Lethal Injections Affirmed. [online] Available from: http://cen.acs.org/articles/93/i27/Sedative-Lethal-Injections-Affirmed.html

Cover Image Credit: Thomas Boyd (The Oregonian)

Diatoms and Death By Drowning

Diatoms and Death By Drowning

A body is found floating in a lake, the circumstances surrounding the death a complete mystery.

One might assume the cause of death to be drowning, and for this there may be certain pathological indicators. But failing these indicators, how can you be so sure that the victim drowned? It could be that they were killed elsewhere, their body tossed into the lake to eliminate suspicion. Or perhaps they did drown, but in alternative circumstances in another body of water. The scenarios are endless. But how can these questions be answered?

The key to this problem might just be a diverse group of microscopic algal organisms known as diatoms.

Dia

Perhaps you’ve heard of them. These asexually-reproducing organisms exist in a vast variety of shapes, sizes and colours, plentiful in many aquatic environments and existing in a tremendous range of populations. A particularly important feature of diatoms is their silica-based cell wall, producing an especially distinctive appearance that can vary greatly between different species. This cell wall enables diatoms to be particularly resistant to decay, so they may persist in an environment for a long time. It is their abundance, uniqueness and resistance that has allowed diatoms to be of such great use in the field of forensic limnology, that is the study of freshwater ecology in a legal context.

So how can these minute microorganisms help determine the circumstances surrounding a suspicious death?

Imagine a person drowning. As the head is submerged, water is inhaled into the lungs, along with any microorganisms contained in that water. In this case, diatoms. So the presence of diatoms in the lungs proves death by drowning? Not at all. Water can passively reach the lungs regardless of whether the victim was dead or alive by the time they reached the water. However if the victim is alive, when diatoms hosted by the water reach the lungs, they will be circulated around the body via the bloodstream, being deposited in different bodily tissues and internal organs.

So with this in mind, the investigator may be able to conclude that cause of death is likely to have been drowning if these diatoms are detected in the internal organs. At this point it is necessary to note that diatoms may already exist within the body, as these algal communities are found in various environments other than water. It is therefore necessary to establish a kind of match between the diatoms in the suspected drowning medium and those inside the body of the deceased. By studying the species of diatoms present and their abundance, it may be possible to conclude whether the diatom populations are consistent with one another. Interestingly diatom populations can also vary seasonally, thus may be able to provide some insight into the time of year in which a victim drowned based on the diatoms extracted from the remains. Comparisons such as these can be made by collecting water samples and extracting diatoms from bodily tissues and internal organs (often through acid extraction), before comparing the diatoms using light or electron microscopy.

The possible applications of the study of diatoms is by no means limited to these scenarios.

Dia2

Numerous features of diatoms make these microorganisms an ideal focus for analysis in forensic investigations. Their minute size means that they can be readily transferred from the crime scene by objects or people, with perpetrators unlikely to be aware of the presence of these organisms. The resilience brought about by the silica-based cell wall allows for them to persist in the human body even beyond later stages of decomposition, during which time cause of death by pathological means may be more difficult. The distinctive morphology of diatoms allows for species to be distinguished from one another, and their abundance and variation results in different bodies of water developing very distinctive assemblages of diatoms.

Unfortunately the use of diatoms as an indicator of cause of death by drowning is somewhat controversial, highlighting the need for further research in this area of study.

References

Horton, B. P. Boreham, S. Hillier, C. The development and application of a diatom-based quantitative reconstruction technique in forensic science. 2006. University of Pennsylvania Scholarly Commons.

Krstic, S. Duma, A. Janevska, B. Levkov, Z. Nikolova, K. Noveska, M. Diatoms in forensic expertise of drowning – a Macedonian experience. For Sci Int. 127(2002), pp. 198-203.

Pollanen, M. S. Diatoms and homicide. For Sci Int. 91(1998), pp. 29-34.

Scott, R. S. Morgan, R. M. Jones, V. J. Cameron, N. G. The transferability of diatoms to clothing and the methods appropriate for their collection and analysis in forensic geoscience. 241(2014), pp. 127-137.

Featured Image – https://www.flickr.com/photos/carolinabio/6622267417

From Mummies to Grave Wax – The Preservation of Human Cadavers

From Mummies to Grave Wax – The Preservation of Human Cadavers

Warning: Graphic images included.

When we envisage the decomposition of a corpse, the images that probably come to mind are of a rapidly-decayed, foul-smelling body quickly turning to sludge and bones. But there are actually other pathways a body can take following death, some of which can be of great importance in a forensic investigation. I’m namely talking about mummification and adipocere.

Mummification

Thanks to the Ancient Egyptians, we all know about mummification, a particular process of body preservation. When these guys mummified their dead, this routine typically involved the removal of the internal organs, most notably the brain, which was pulled out through the nostrils. Finally, the body would be wrapped in linens and salt and left to dry. The end result would be a remarkably well-preserved body displaying features that would have usually been lost to decomposition.

Aside from this especially famous post-mortem ritual, there are actually a number of ways by which a body can become mummified. For instance, more modern intentional mummification would utilise chemicals to preserve the body. But how can mummification occur naturally, and what are the implications of this in a forensic investigation?

The natural preservation of a cadaver is highly dependent on the surrounding environment, with only very specific conditions causing the body to mummify. A range of factors can play a part in this phenomenon, including temperature, humidity and the action of bacteria and other microorganisms.

In hot, dry climates moisture can evaporate from the skin at such an accelerated rate that the process of mummification can occur. As the skin is rapidly dehydrated, it often takes on a dark and leathery appearance. The internal organs may be preserved to an extent, though will typically undergo some level of decomposition so are at least likely to be smaller in size. Hot, dry climates can also hinder bacterial growth, limiting the bacterial decay and further preserving the body. Hot, sandy deserts are perhaps amongst the first scenarios that come to mind when considering mummification, but mummified remains have also be discovered in attics, basements, and even within the walls of buildings.

Conversely, especially cold and dry environments can also bring about mummification. The cold temperatures can significantly slow microorganism activity, once again reducing the rate of decomposition and aiding in preservation. A famous example of this is the natural mummy Otzi the Ice Man, believed to have died thousands of years ago but preserved through mummification induced by extremely cold temperatures.

mumm1

Mummifying conditions are not limited to temperature-based factors. Environments of extreme salinity (salt content) can preserve cadavers. The majority of bacteria cannot survive in highly salty conditions, thus severely reducing microbial action on the body. Furthermore salt itself acts as a desiccant on the soft tissue, dehydrating the body and drawing out water much as high temperatures would. An example of this type of mummification was experienced in Iran, where a number of mummies were found in the Chehrabad salt mines.

Mummified remains have also been found in bogs or marshland, in which the excess water, organic material and anaerobic (oxygen-free) environment prevents a great deal of bacterial action, thus preserving the body. This is something of a contrast to the typical hot, arid conditions mostly associated with mummified remains, but bogs with particularly acidic water, low temperatures and a lack of oxygen can essentially pickle a body. Thousands of these “bog bodies” have been recovered over the years, perhaps the most famous being the Lindow Man, determined to be the victim of a prehistoric ritual killing.

The conditions described may not necessarily induce mummification throughout the entire cadaver, but in some cases may cause localised mummification, if only particular areas of the body are exposed to these conditions. Mummification most often occurs in the face, scalp, chest and back, but typically begins in the extremities such as the fingers and toes.

Adipocere

Another phenomenon that can assist in preservation of a cadaver is the formation of adipocere. Known as “grave wax”, this is a soapy, white or grey wax composed primarily of saturated fatty acids such as palmitic and stearic acid formed through the hydrolysis and hydrogentation of body fats (Forbes et al, 2005). Numerous theories have been put forward to suggest how adipocere forms, namely the saponification, hydrogenation and fat migration theories.

A cadaver presenting adipocere. Credit: Kumar et al, 2009

The type of environment required for adipocere formation is somewhat different from that suitable for mummification. It is often encountered in especially humid graves with little or no air access, thus oxygen-poor, such as a bog or certain bodies of water. The formation of adipocere can take weeks if not years to form depending on the climatic conditions, the rate at which it forms being further affected by the environment and circumstances surrounding the cadaver. Depending on the extent to which it forms, adipocere can produce a waxy layer across the body and act as a barrier against the usual process of decomposition, providing significant protection over time as adipocere itself is fairly resistant to decay.

So these are some of the alternative routes of decay a body can take post-mortem. But what does this mean to the forensic scientist?

In some cases, the occurrence of mummification or adipocere formation can be of assistance to a forensic investigation, as it may be possible that certain aspects of the deceased person’s appearance and even any injuries they might have acquired will be preserved. Mummified tissues can even be rehydrated to aid in visualising injuries and other distinguishing marks. Similarly, the formation of adipocere can preserve tissues and organs along with recognisable facial characteristics. This can in theory aid in identification if the victim is unknown or even determining cause of death.

Furthermore, just as the stage of decomposition of a body can roughly indicate the post-mortem interval (time since death), mummification and adipocere can provide some indication in that a certain amount of time is required for mummification to occur. Approximately 6-12 months are required for the natural mummification of an adult, with a child’s body requiring less time to become mummified (Gitto et al, 2015), though in some cases mummification has been reported in a matter of weeks or even days (Sledzik and Micozzi, 1997). Of course these time periods can vary widely depending on climatic conditions and a number of other factors, but they may provide assistance nonetheless. To an extent it may be possible to determine the rough age of the remains based on the weight of the mummified cadaver, as more recent bodies will be heavier than those which are older and have lost a greater proportion of water content.

So given the right conditions, processes such as mummification and adipocere formation can interestingly be a great aid to the forensic investigator.

References

Bereuter, M. T. L. Lorbeer, E. Reiter, C. Seidler, H. Unterdorfer, H. Post-morten alterations of human lipids – part I: evaluation of adipocere formation and mummification by desiccation. Human Mummies. 3 (1996), pp. 265-273.

Bryd, J. H. Castner, J. L. 2010. Forensic Entomology: The Utility of Arthropods in Legal Investigations. Boca Raton, Florida: CRC Press.

Forbes, S. L. Bent, B. B. Stuart, H. The effect of soil type on adipocere formation. For Sci Int. 154 (2005), pp. 35-43.

Gitto, L. Bonaccorso, L. Maiese, A. dell’Aquila, M. Arena, V. Bolino, G. A scream from the past: A multidisciplinary approach in a concealment of a corpse found mummified. Journal of Forensic and Legal Medicine. 32 (2015), pp. 53-58.

Kumar, T. S. M. et al. Early adipocere formation: A case report and review of literature. Journal of Forensic and Legal Medicine. 16 (2009), pp. 475-477.

Rich, J. Dead, D. E. Powers, R. H. 2005. Forensic Medicine of the Lower Extremity. Totowa, New Jersey: Humana Press Inc.