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Can Cuticle Compounds Be Extracted From Insects Preserved in Ethanol?

Polybia paulista wasp nest

Polybia paulista is a species of swarm-forming paper wasp, a nest of which is shown here. A new study by researchers at the Universidade Estadual de Mato Grosso do Sul in Brazil found that chemical compounds from P. paulista cuticles could be extracted and analyzed from specimens preserved in ethanol, which has typically not been viewed as an ideal solvent for extracting cuticular compounds. The finding raises the possibility that ethanol-preserved insects can possibly be used for the analysis of cuticular chemical compounds (CHCs), allowing the use of a single sample for both CHCs analyses and other studies, such as DNA sequencing. (Photo by Fabio Seitoku Nagamine, via Wikimedia Commons, CC BY-SA 4.0)

By John P. Roche, Ph.D.

Insects have many chemical compounds in their outer cuticle that perform a range of functions, including protection, water balance, temperature balance, and communication. Because of the biological importance of these cuticular compounds, scientists often study which chemicals occur in species. To do so, investigators often use non-polar solvents such as hexane to extract compounds from the cuticle, and then they analyze the extracted chemicals using gas chromatography.

Insects are often preserved in ethanol, which is an excellent preservative, but it is generally not viewed as an ideal solvent for extracting cuticular compounds. However, a new study published this month in Environmental Entomology revisited this assumption to see if chemicals from insect cuticles could indeed be extracted and analyzed from insects preserved in ethanol. The study, conducted by Dayana Alves da Silva Cunha and colleagues at Universidade Estadual de Mato Grosso do Sul in Brazil, used a social wasp species that lives in South America, Polybia paulista.

A swarm-forming paper wasp, P. paulista is famous for its venom, which is being explored as a powerful anti-cancer therapy. But P. paulista is also a fascinating subject for work on chemical ecology of cuticlar compounds in wasps.

The cuticle of an insect is the outer layer of the exoskeleton. It consists of a thin, waxy outer ectocuticle and a thicker procuticle. The best-known chemical in the cuticle is a polysaccharide called chitin, which is found in the procuticle.

Cuticular compounds include hydrocarbons, fatty acids, and proteins. Hydrocarbons are non-polar molecules consisting of only hydrogen and carbon. They come in linear or branched chains with backbones of linked carbon atoms, and they can serve a variety of functions. Some hydrocarbons contain all single bonds between their carbon atoms, while others have one, two, or three double carbon bonds. Hydrocarbons with all single bonds often function to prevent desiccation in insects. Hydrocarbons with double bonds can form much more complex structures and are often involved in insect communication. Examples of research uses for cuticular hydrocarbons (CHCs) in insects include use as taxonomic markers and use for studies of biogeography.

To examine if cuticular compounds could be removed from wasps preserved in alcohol, Alves da Silva Cunha and colleagues froze five colonies of swarm-forming paper wasps gathered from the field in Brazil. In one experiment, they immersed female wasps in the solvent hexane, extracted the hexane, and analyzed the chemicals in the hexane using gas chromatography. Then they took female wasps that had been earlier immersed in hexane and soaked them in ethanol for 30 days. Next, they added hexane again, extracted the hexane, and analyzed the chemicals in the hexane using gas chromatography.

In a second experiment, the researchers immersed female wasps in ethanol for 30 days and then immersed them in hexane and extracted chemicals for gas chromatography. They also tested how many chemicals were found in the ethanol in which the wasps had been soaked.

The results of the first experiment showed that in wasps that had not yet been soaked in ethanol, they found 54 compounds (48 of these were hydrocarbons and three were fatty acids). In wasps that had been soaked in ethanol, they found 52 compounds (47 of these were hydrocarbons and three were fatty acids).

Importantly, their statistical analysis found no significant difference in the composition of cuticular compounds found in the cuticles of wasps preserved in ethanol compared to those that were not preserved in ethanol.

In the second experiment, in the wasps soaked in ethanol for 30 days, they found 48 hydrocarbons and three fatty acids, with a total of 54 compounds. In the analysis of the ethanol itself in which the wasps were soaked, they found 49 hydrocarbons and three fatty acids, with a total of 54 compounds.

Thus, they discovered that wasps preserved in ethanol yielded very similar chemical profiles to wasp specimens not preserved in ethanol, and that insects preserved in ethanol could be used for extraction studies of cuticular chemical profiles.

Alves da Silva Cunha and colleagues concluded, “Even ethanol-preserved insects used in other biological studies, especially those involving DNA extraction, possibly can also be used for the analysis of cuticular chemical compounds, thus allowing the use of the same sample for DNA sequence and CHCs analyses.”

This research focused on one set of parameters in one wasp species. The research team points out that additional studies are needed to examine outcomes of extraction of cuticular compounds using different ethanol concentrations, different durations of ethanol contact, different storage temperatures, and different species of insect. But these findings provide an important reference point and a valuable comparison for other studies.

John P. Roche, Ph.D., is an author, biologist, and educator dedicated to making rigorous science clear and accessible. Director of Science View Productions and Adjunct Professor at the College of the Holy Cross, Dr. Roche has published over 200 articles and has written and taught extensively about science. For more information, visit https://authorjohnproche.com/.

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