Blow flies, in the family Calliphoridae, such as the Calliphora vicina adult shown here, are active decomposers and thus important to forensic entomology investigations, as they will find and lay eggs on a corpse in short order. Identifying the species of blow flies present is a critical step in such investigations, but it typically requires rearing collected fly larvae to adults first. However, a new “real time” method for conducting mass spectrometry could allow maggot specimens to be analyzed and identified in a matter of minutes—even up to six species at a time from the same sample. (Photo by Susan Ellis, Bugwood.org)

By Ed Ricciuti

Ed Ricciuti

It’s an almost impossible task, surely not for the squeamish. When forensic entomologists investigate an untimely death, they often must sort look-alike species of blow fly maggots in a squirmy, mucky mass, collected from thousands of them that have been noshing on a dead body. These maggots sampled from a corpse can provide forensic investigators with a host of clues to the circumstances of death, but the precise details depend upon identifying species that are collected.

Trouble is, all blow flies belong to the same taxonomic family (Calliphoridae), and the maggot, or larva, of one species is pretty much a carbon copy of any other, to the eye at least. Telling them apart is so difficult that forensic entomologists typically raise them through their three instars, or larval stages, to adults, which are more easily differentiated—but this takes considerable effort, resources, and time.

Rabi Musah, Ph.D.

From now on, the wait may be over. Scientists at the University at Albany have combined a quick-time mass spectrometry method with an innovative statistical model that makes the job of identification much faster and easier than any previous techniques. The researchers, led by Rabi Musah, Ph.D., professor of chemistry at the University at Albany, describe the problem and their proposed solution in a report published in late February in the journal Analytical Chemistry.

“Since knowledge of the species of insect is crucial to being able to glean useful information about infestation, an approach to the determination of the identities of the species present in mixtures is of high interest,” Musah and colleagues write. “When entomological samples are procured, they are usually submerged in aqueous ethanol for storage, often in the form of multiples of eggs, larvae, or pupae, which can make it challenging to accurately determine the identities of the species that are present.”

Using a mass spectrometry technique developed about 15 years ago that is highly versatile, the research team analyzed a soup of mixed maggots in a water-ethanol suspension and recorded the spectral signatures of chemicals shared by and unique to up to a half-dozen different species. They developed an innovative statistical model that, when applied to the spectrometric data, easily differentiated the species in mixtures ranging from two to six of them.

“We have long been interested in developing approaches to make crime-scene analysis easier and more rapid, and the ability to apply mass spectrometry and machine-learning approaches to help to resolve challenges in forensics was a natural fit for what we do,” says Musah.

Typically, when maggots are collected from a corpse by a forensic entomologist, some are killed by immersion in water just below boiling and stored in a mix of water and ethanol. The hot water treatment is necessary because, if placed directly in preservative, maggots shrink, which could make them appear younger than their true age. About half the maggots usually are kept alive for further investigation and rearing to adulthood for identification, which is so critical for reconstructing events surrounding the death.

A technique called direct analysis in real time (DART) mass spectrometry allows for analysis of a sample nearly instantaneously and without special preparation. The operator simply places the sample on a holder in the open air between the DART ion source and the spectrometer. (A beetle is shown in the image here.) A new report in the journal Analytical Chemistry shows the technique’s potential application in forensic entomology investigations, with researchers successfully analyzing and identifying the species of maggot specimens in a matter of minutes—even up to six species at a time in the same sample. (Photo by Rabi Musah, Ph.D.)

Why is knowing species so important? One example is that, if a species that prefers wet habitat is found on a corpse in dry surroundings, chances are the body was moved after death. Species can also indicate the time of death, since some adult females arrive at a corpse and lay eggs within minutes after death, while others arrive later.

The faster forensic scientists identify the species, therefore, the quicker the investigation can progress. The key to the process developed at Albany is the use of a technique called direct analysis in real time (DART) mass spectrometry. It analyzes a sample instantaneously, making traditional, time-consuming mass spectrometry look plodding.

Mass spectrometry, developed early in the last century, is an old standby for detecting the kind and amount of chemical components of a sample by separating them into gaseous ions and measuring their mass-to-charge ratio, which is then plotted on a mass spectrum. The ions, and thus the chemical composition of the sample, can then be identified.

Traditionally, ionization occurs in a high vacuum chamber, which can make introduction of samples difficult and somewhat limits the variety of compounds that can be screened. Some materials cannot be converted to the required gaseous state. Newer techniques can be performed under normal atmospheric pressure but involve high temperatures or radiation that require shielding to protect the operator and still require elaborate preparation of the sample. DART, on the other hand, does its job in open air at normal atmospheric pressure and can analyze samples as they are without special preparation. The operator merely places the sample on a holder in the open air between the DART ion source and the orifice of the spectrometer, rather than inside it.

“The ease and rapidity of the technique, the fact that samples can be analyzed as they are in their original form, and the range of compounds that can be detected make DART mass spectrometry an extremely powerful technique with numerous applications in forensic science,” says Musah.

In Musah’s team’s study, the data obtained using DART mass spectrometry was then subjected to advanced statistical analysis to create a predictive model, which was then applied to a statistical model, enabling the researchers to distinguish between different species in the ethanol suspension and even identify their different life stages. The model used conformal prediction, a relatively new statistical tool in machine learning that uses past experience to predict future performance.

The Albany researchers are carrying on a tradition started in the 13th century by Chinese physician Song Ci. After a man was slain by slashes of a sickle, farmers in the locale were told to bring in their sickles for examination. Only one attracted blow flies, drawn by traces of blood and tissue on the blade. Its owner confessed, resolving the first known criminal case that employed forensic entomology.

Ed Ricciuti is a journalist, author, and naturalist who has been writing for more than a half century. His latest book is called Bears in the Backyard: Big Animals, Sprawling Suburbs, and the New Urban Jungle (Countryman Press, June 2014). His assignments have taken him around the world. He specializes in nature, science, conservation issues, and law enforcement. A former curator at the New York Zoological Society, and now at the Wildlife Conservation Society, he may be the only man ever bitten by a coatimundi on Manhattan’s 57th Street.