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.

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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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Interview with Forensic Entomologist Susan Gruner

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What is your expertise within forensic science and what does this type of job role typically involve?

Forensic entomology can be divided into three groups: stored-product, urban and medico-legal (or medico-criminal) entomology. I specialize in the field of medicolegal forensic entomology. Generally speaking, forensic entomologists can estimate a postmortem interval (PMI) or time since death based upon the presence (or lack thereof) of insects collected on or near a body. Certain families of insects arrive in a predictable manner on corpses, especially the calliphorid flies (blow flies). Based upon the ages of specimens collected, a PMI can be estimated. Age of the specimens is directly related to the temperatures that the insects are experiencing; the higher the temperature (obviously to a certain limit), the faster they grow.

In a best-case scenario, when a body is found, a forensic entomologist should be called and he or she would collect and process the insect evidence. The second best scenario would be that properly trained law enforcement personnel would collect the insect evidence and then send it to a forensic entomologist for processing.

On occasion, forensic entomologists have to testify in court, just as would any other expert witness. The testimony usually focuses on time of death, but not always. Sometimes a body may have been moved and the calliphorids present can sometimes help determine such a thing. There was a case where a woman was murdered in a parking lot in the city of Jacksonville (Duval County). Her body was dumped in rural Clay County, but not before those city flies had a chance to deposit eggs on her body. The trial was held in Duval County.

Calliphorid female flies are attracted to areas of trauma on a body, where they will deposit eggs. Sometimes the trauma is not noticeable to the naked eye, but can be determined during autopsy. But forensic entomologists know that flies congregate and deposit eggs on areas of trauma on a body.

In September 2006, I received a call from the Jacksonville Sheriff’s Office that the body of an elderly man had been found in a culvert off of I-10, a major highway. I did not know what a culvert was until this case (for those who do not know, it is a tunnel that carries water under a road). It was necessary to climb down a steep angled wall to make an entomological collection. When I reached the body, I noticed that the man had hundreds of calliphorid larvae around his neck despite a lack of visible wounds or bruised skin. I told the detectives that the medical examiner would find the cause of death to be strangulation. I got a lot of strange looks from the detectives that day, but the autopsy showed the man had indeed been strangled to death.

I guess you could say that being a forensic entomologist requires getting very dirty, too. The first time I ever returned from a death scene, my husband had to have the car detailed because it smelled like human decomposition. But as horrific as it sounds, I continue to be fascinated by what calliphorid larvae can reveal about death and decomposition and how they can help solve crimes.

What led you to become involved in forensic entomology?

I thought I wanted to be a veterinarian, and one of the ways to get into vet school is to major in entomology. On the first day of my first entomology class, the professor handed out an article about the Body Farm in Tennessee. I changed my mind about wanting to be a veterinarian that instant.

What in particular was the focus of your PhD research in forensic entomology?

The main focus of my doctoral research was to study the life cycle of the forensically important calliphorid, Chrysomya megacephala (the oriental latrine fly). The main focus of my research was to study their development rates at different ambient temperatures.

Historically, calliphorid development rates have been studied by placing a few hundred calliphorid eggs in a cup (or cups) in a rearing chamber set to a certain temperature. The time is monitored as the larvae grow through three larval stages, a puparial stage, and then emerge as adults.

But the problem with testing in this manner is that calliphorid masses generate heat. Even a small amount of maggots can generate heat above ambient, thus the set chamber temperature is not really the temperature that the growing larvae/pupae are experiencing. For the development research, I used 10 larvae per container; an amount not capable of generating heat above ambient temperature.

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Liver is a standard feeding substrate for calliphorid larvae in colony.  But the small amount of liver needed for only 10 larvae per Petri dish dried up quickly, killing the larvae, so I developed a liver agar feeding substrate that would not dry up1. Then I was able to study precisely how the larvae developed at eight different temperatures.

This was grueling work in which larvae were checked at 4-hour intervals 24/7 for most of the duration of the development research, which took 15 months. As I had no funding to hire anyone to help me, I trained my husband to do it. He took the day shift, going back and forth to the lab every 4 hours as I tried to get some sleep. I spent nights (from 9 PM to 9 AM) in the lab. I was so sleep-deprived that it would have been dangerous for me to drive back and forth during the middle of the night.

There were times when we had to examine every larva individually to determine stage. This required looking at the anal spiracles of a larva under a microscope with a gentle but steady hand. During all those months of research when we were sleep-deprived and miserable, we never squished or killed a larva. I am very proud of that.

I also studied different size maggot masses and their respective temperatures as they grew in age, size and volume. And finally, I studied different models that could potentially be used to calculate age of a larva (or larvae). Most forensic entomologists use a linear model to estimate a PMI, but insect development is not linear, especially that of calliphorids.

Do you believe there are any common misconceptions surrounding this field of work?

There are too many to list, but I will name a few.

  1. It is nothing even remotely similar to anything seen on TV.
  2. Sadly, I know of only one person who is a full-time forensic entomologist in the USA (in Texas). Most who received their M.S. and PhDs studying forensic entomology are not teaching or practicing forensic entomology. They may take a case or testify in court on occasion, but that is rare.
  3. Despite the potential importance of collecting insect evidence at death scenes, it is not done. The vast majority of detectives, crime scene technicians, etc., have no idea how to collect insect evidence and forensic entomologists rarely get called to process the insect evidence at death scenes.

Do you think that there are any significant gaps in research in forensic entomology?

Yes. I would say the biggest problem in this field is the poor quality of research, especially when it comes to studies regarding development rates of forensically important insects. Most (almost 80%) of the manuscripts published in peer-reviewed journals in the past 30 years lack replication and have poor experimental design. This is an embarrassment to the science and needs to change. But, there are many dedicated scientists in this field who are trying to do good work.

Finally, how do you feel about the extent to which forensic entomology is harnessed in legal investigations?

Forensic entomology is overlooked much of the time. Collection of insect evidence is not difficult, but as I mentioned above, such evidence is rarely–if ever–collected.

Twitter: https://twitter.com/Entomophila

 

If you’re a forensic scientist (academic or industry) or a crime scene investigator and would like to be part of this series of interviews, get in touch by emailing locardslabblog[at]gmail.com.

This is Part 6 of our series of interviews with forensic professionals.

Interview Series Part 1 – Interview with Forensic Identification Scientist Alexandre Beaudoin

Interview Series Part 2 – Interview with Forensic Expert Robert Green OBE

Interview Series Part 3 – Interview with Forensic Expert & Consultant Gareth Bryon

Interview Series Part 4 – Interview with Forensic Identification Specialist Donna Brandelli

Interview Series Part 5 – Interview with Forensic Video Analyst David Spreadborough

Interview Series Part 6 – Interview with Forensic Accountant Sundaraparipurnan Narayanan

Identifying Insects with Spectroscopy

Identifying Insects with Spectroscopy

Entomology, that is the study of insects, can provide vital information during a forensic investigation. After an individual dies their body begins to undergo a complex decomposition process almost immediately, attracting a variety of insects along the way who wish to colonise, feed on the temptingly putrefying remains and reproduce.

Specialists have been taking advantage of this fact for hundreds of years, allowing us to discover that the types of insects present on a cadaver and the age of these insects can prove invaluable in estimating how much time has passed since the victim died (known as the post-mortem interval). Simply put, certain species prefer the decomposing corpse at different stages in the decay process, and with the right information, investigators can study the insects and their ages and begin to develop a kind of timeline.

Currently, accurately identifying species and establishing the development stage of an insect can be time-consuming and requires the expertise of an entomologist and potentially DNA analysis. This is obviously not ideal – your average police force does not have an entomologist on hand, nor do they have oodles of times to dedicate to insect identification. Even with the assistance of an entomologist, accurately determining the age of maggots can be problematic. Although larvae may be of a certain age, their length and weight can be affected by a variety of factors that may not be accounted for, such as starvation (Singh and Bala, 2009).

As you might expect, researchers are searching for ways to resolve this issue, and analytical chemistry might just be the answer.

As analytical chemistry progresses and increasingly advanced analytical techniques are developed, we are seeing more and more fascinating applications of these instruments to established areas of study. In a recent study published in Forensic Science International, researchers took a well-established technique and applied it to forensic entomology. In this case, they used a form of infrared spectroscopy.

Infrared spectroscopy is an analytical technique which determines the amount of radiation absorbed by a molecule. Infrared light is directed towards to sample and, depending on the molecule, a certain amount of radiation will pass through the sample and some will be absorbed.  When a molecule absorbs radiation, the bonds within it begin to vibrate. Different bonds will vibrate and be influenced by surrounding atoms to a different extent, thus allowing for a unique ‘spectrum’ to be produced. This spectrum is essentially a graph displaying how much radiation was absorbed by the sample at what wavelength. Scrutinising this spectrum can allow the analyst to determine what kind of molecules are present. Although this is not sufficient to specifically identify compounds, the spectrum produced can at least be used to distinguish between different samples, which will produce different spectra. The spectra essentially act as ‘fingerprints’ for different substances.

FTIRSpectrum

Typical IR spectroscopy spectra.

If you want to know a bit more about this technique, Compound Chemistry has a great little page on IR spectroscopy.

So back to how this analytical technique can be useful in forensic entomology. The proof of principle study to which I’m specifically referring aimed to both identify the species of larvae and the life cycle stage using vibrational spectroscopy, in this case Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. A slightly long-winded name, but in short this is simply a form of IR spectroscopy that allows in situ analysis of solid or liquid samples without the need for sample preparation. Anyone who has spent many painful hours preparing samples for analysis will appreciate the benefit of this.

Three species commonly encountered at incident scenes were used in the study; C. vomitoria, L. sericata, and M. domestica (that is the bluebottle fly, the green bottle fly and the common housefly respectively). One of these species (the C. vomitoria) was also selected for a study focussing on the life cycle, in which spectra were collected for each time point in the insect’s life cycle. Scans were based on a crushed mixture of epidermis and internal matter (not possible for a ‘no maggots were harmed during the making of’ notice then). The results were promising, indicating FTIR spectroscopy could be a great tool in forensic entomology.

But surely there is a whole range of analytical instruments out there (yes, there sure is), so why would this one be any more suitable for forensic entomology? One of the major benefits of FTIR is the possibility of handheld IR instrumentation, which basically means it can be used in situ at the scene of a crime or other incident. Granted the investigator would need the appropriate equipment, but it beats shipping samples back to the lab and waiting for analysis. IR spectroscopy is a non-destructive technique (okay, the insects were somewhat mutilated in this study, but nevertheless the samples themselves remained after analysis). The ability to perform analyses without destroying the sample has a huge benefit, particularly if the available sample is limited, allowing for alternative tests and future analysis to be conducted if necessary. This of course is an advantage in forensic science. Also of great benefit to a legal investigation, IR instrumentation is fast, with spectra being collected in a matter of minutes.

There is however the glaring problem of the cost of analytical instrumentation. As I previously stated, your average police force may not have a forensic entomologist on hand… they equally may not have the funds to purchase analytical instrumentation such as IR spectrometers.

Bearing in mind this was merely a pilot study, using a very limited sample size, the research shows some promising results – that it is possible to classify species and life cycle stage using IR spectroscopy. Were this to be expanded upon, you could theoretically develop a database of IR spectra collected from different species of insects at different stages of development, allowing future spectra obtained from unknowns to be compared and, hopefully, identified.

References

Pickering, C. L. Hands, J. R. Fullwod, L. M. Smith, J. A. Baker, M. J. Rapid discrimination of maggots utilising ATR-FTIR spectroscopy. Forensic Sci Int. 249 (2015), pp 189-196.

Singh, D. Bala, M. The effect of starvation on the larval behaviour of two forensically important species of blow flies (Diptera: Calliphoridae). Forensic Sci Int. 193 (2009), pp. 118-121.

Bugs, Bleach and Bodies

Bugs, Bleach and Bodies

Following on from my last post focusing on forensic entomologist Dr Zak, it seemed apt to carry on with a recent piece of forensic entomology research I came across.

In forensic entomology, post-mortem interval (the time since the victim’s death) is typically estimated based on the types of insect present at the scene and, most importantly, their stage of development. It is probably of no surprise that flies (Order: Diptera) play a big part in insect colonisation of a cadaver, thus have been subject to a lot of research in forensic entomology. The life cycles of certain flies are relatively well known in terms of the different stages of development and when those stages are likely to be reached.

fly2The lifecycle of a fly consists of a number of stages: egg, 1st instar, 2nd instar, 3rd instar, pre-pupa, pupa, and finally the adult fly (instar refers to stages of moulting as larvae). The time taken to reach each phase can vary between species. And there are of course factors which affect these development times, some that have been greatly studied, including environmental temperature, sun exposure, food availability, and even drugs taken by the deceased prior to death.

Now researchers are branching out into the study of other affecting factors, in this case typical household products. It isn’t uncommon for certain products to be spilled or in some other way present at the scene of a death (whether criminal or otherwise). Maybe a victim was smothered in acid in vain attempts to dispose of the body (good luck with that one) or perhaps the deceased happened to have slathered on some insect repellent immediately before his or her untimely death. Regardless, the ways in which chemicals appear at a death scene are plentiful, and they need to be taken into account if we’re relying on a somewhat environmentally-dependent factor to determine the post-mortem interval.

Enter researchers at the University of Lille Nord de France.

These guys and girls aimed to figure out how some common chemicals might affect the development of a particular species of common fly (in this case Lucilia sericata, the green bottle fly) by allowing the first instar larvae to feast on beef liver laced with different chemicals – in particular bleach, perfume, hydrochloric acid, caustic soda, insecticide, mosquito repellent, and gasoline. Specimens were subjected to either low concentrations of the chemicals (supposedly the equivalent to a realistic quantity being splashed on or otherwise applied to the body) or high concentrations. The development of the different groups of Lucilia sericata were then studied, allowing researchers to establish whether the chemicals present delayed, accelerated or had no effect on larval development, as well as possible effects on insect size, survival rate and sex ratio.

The results were interesting even if they were not wildly significant (ignoring the chemicals which just outright killed all subjects, not making them terribly useful post-mortem indicators). Low concentrations of mosquito repellent and caustic soda extended the development time of the larvae (361 hours and 352 hours respectively, in comparison to the control of 333 hours), as did high concentrations of perfume (342 hours). These figures I’ve listed are hours taken to reach adulthood, the mean values being used. At first glance these may not seem like such large differences, but what a difference a day makes when trying to pinpoint time since death (though the confidence interval typically used by entomologists is about 25 hours). There were also certain size variations noticed between adults fed on different sources, though this was not a particular focus of the study so no conclusions can really be made. The research also looked at survival rates and sex ratio, but I will skim over this (with the exception of pointing out that perfumed meat resulted in the survival of more females over males – we ladies do enjoy a good perfume!). Despite the limitations of the work and the relatively small differences caused, differences were observed, which suggests this path of research could be a fascinating and relevant one.

Realistically this was a pretty limited study, looking at a single species of laboratory-reared larvae and examining a small handful of household products, but the results are interesting nonetheless, indicating the impact of household chemicals on necrophagous fly development. We know conclusively that certain factors can have a renowned effect on the development of insects, thus affecting what we know about figuring out time since death. However taking into account what else might be on the victim’s body is something that may be overlooked, or at least not considered by the lay person perhaps.

So next time you’re slapping on some repellent to keep the mosquitos at bay, give a thought to the forensic entomologist whose day you might be making a little more tricky.

References

Aubernon, C et al. In vitro effects of household products on Calliphoridae larvae development: implication for forensic entomology. J Forensic Sci. 60 (2015), pp 226-232.

 

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Scientist Special: Zakaria Erzinçlioglu

Scientist Special: Zakaria Erzinçlioglu

From fly-infested corpses to forensic politics, self-titled “maggotologist” Zakaria Erzinçlioglu pioneered forensic entomology in Britain and fought for a better criminal justice system.

Perhaps Britain’s leading forensic entomologist, Zakaria Erzinçlioglu dedicated most of his career to the application of his extensive knowledge of insects to legal investigations. Born in Hungary in 1951, he went on the live in Egypt, the Sudan and finally England, where he carried out much of his work. Most commonly referred to as Dr Zak (I will take a wild guess and assume his colleagues and students struggled with his surname!), he fell into the fascinating world of forensic science somewhat incidentally. As an entomologist with a great interest in the transmission of diseases by insects, in the early 1970s he soon found himself being called upon by the police to offer expert advice on insect evidence. As more and more requests for advice such as this were made, Dr Zak soon realised that this fascinating branch of entomology was where his career would take him.

After dedicating his doctorate to the study of blowfly development, he moved around the country working for a number of institutions, working within the Zoology department at the University of Cambridge, carrying out forensic entomological research funded by the Home Office, and acting as director of Durham University’s Forensic Science Research Centre.

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He contributed his expertise to a range of infamous criminal investigations, including the investigation of a notorious paedophile ring responsible for the death of a 14-year old boy, the trial of serial rapist and murderer Robert Black, and the case of lecturer-turned-murderer Dr Samson Perera. In the investigation involving Dr Perera, a dental biologist suspected of murdering his adopted daughter Nilanthie, Dr Zak was called upon when a number of bones were found in the lecturer’s home and laboratory. Whereas Perera insisted the bones were specimens for medical research (as dental biologist of course often require femurs and spinal cords for their work…), Erzinçlioglu was able to conclude that the bones were recent and had been dismembered, all through the examination of a specific fly still present on some of the samples. Erzinçlioglu’s expertise helped put to rest scores of criminal cases, however his contribution to the field of forensics was not just entomology-based, but also somewhat political.

Dr Zak developed a certain discontentment with the provision of forensic science services in Britain, a disapproval which he did not keep to himself. He called for a single statutory body of forensic science which answered solely to the judiciary, not to be hired by prosecution or defence, allowing the forensic expert to act as an entirely unbiased expert witness. In an article published in the Contemporary Review in 1998, Erzinçlioglu stressed that any forensic expert hired by either side of the adversarial system used in the UK would be “presented with a line that he is required to support”, encouraging the scientist to protect his or her reputation by offering a good service to the employer, thus jeopardising their ability to be an impartial expert. His determination to raise standards in forensic science were relentless, and he even later went on to offer his expertise to cases of miscarriages of justice, charging no fee and simply wishing to aid those who required his expertise.

He went on to publish a number of papers along with “Maggots, Murder and Men”, a book which provided a fascinating introduction to the science of forensic entomology and a range of case studies. He had numerous other publications in progress, but Dr Zak sadly passed away at the age of 50 on 26th September 2002.

References

Erzinçlioglu, Z (2013). Maggots, Murder, and Men: Memories and Reflections of a Forensic Entomologist. NYC: St. Martin’s Press. 12.

Erzilnclioglu, Z. Reform of forensic science provision. Some basic questions. Science & Justice, 40(2000), pp. 147-149.

Innocent. Science and the law: a cause for concern. [online][Accessed 12 Mar 2015] Available: http://www.innocent.org.uk/misc/cr_erzingclioglu_fss.html

The Telegraph. Zakaria Erzinclioglu. [online][Accessed 12 Mar 2015] Available: http://www.telegraph.co.uk/news/obituaries/1412780/Zakaria-Erzinclioglu.html