In two endangered beetle species that live in the Edwards Aquifer in Texas, males and females are nearly indiscernible. A new study suggests the quickest way to ID males versus females is to shine a light through them, in the lighting setup shown here, to illuminate internal organs that reveal the difference. (Photo originally published in Kosnicky 2019, Journal of Insect Science)

By Andrew Porterfield

Nobody really knows how long the beetles Stygoparnus comalensis and Heterelmis comalensis (sometimes known as the Comal Springs dryopid beetle and the Comal Springs riffle beetle, respectively) have lived in an underground aquifer between Austin and San Antonio, Texas, but the two species are now endangered. Although the 2-millimeter-long cave-dwelling insects managed to survive a severe drought in the mid-1950s, they were declared endangered in 1997.

Andrew Porterfield

The federal protection was triggered by changes to their habitat, called the Edwards Aquifer, which has been subject to pumping to supply San Antonio and other cities with water. The aquifer also has been subjected to pollution and competition from introduced species. In 2013, the U.S. Fish and Wildlife Service (USFWS) expanded critical habitat protection for the beetles to encompass 139 acres of the below-ground aquifer.

While habitat protection is one important tool for preserving endangered animals, another is the development of self-propagating refuges. As part of such efforts, knowing the proportion of males and females is a key component of propagation and performing mating experiments. However, distinguishing the sex of S. and H. comalensis has been challenging.

The sizes between S. comalensis males and females overlap considerably. In addition, males have tufts on their metasternums which females lack, but these features are very difficult to observe. For H. comalensis, females are slightly larger but have no external features that can be used to distinguish between sexes. For both species, poor lighting and viewing could easily lead one to mix up females and males.

Heterelmis comalensis, sometimes known as the Comal Springs riffle beetle, is an endangered species that lives in the Edwards Aquifer in Texas. (Photo credit: Mike Quinn, TexasEnto.net, CC BY-ND-NC 1.0)

To find a reliable way to determine sexual dimorphism in the two beetle species, Ely Kosnicki, Ph.D., a freshwater ecology scientist with BIO-WEST, Inc., an environmental consulting firm in San Marcos, Texas, took measurements of several external features and experimented with a variety of lighting and photographic techniques to determine sex. The results, published in July in the open-access Journal of Insect Science, did not find any helpful measurements of external features but did find that laterally illuminated internal abdominal structures could accurately determine males from females.

Adding to the challenge was the fact that the beetles, especially S. comalensis, are rare and difficult to retrieve. In 2018, the USFWS had just 15 living adults, and Kosnicki’s study looked at nine female and six male specimens preserved by the USFWS.

Kosnicki took the preserved S. comalensis specimens and photographed them with a high-definition color camera, mounted on a stereoscope. Measurements were taken with imaging software. Length and width of ventrites (sternums), elytrons (hardened forewings) and pronotums (thorax plates) were determined. Kosnicki also experimented with different angles of illumination for photographing the beetles.

Under a lateral lighting setup, the female (left) and male (right) of the Stygoparnus comalensis, sometimes known as the Comal Springs dryopid beetle, can be differentiated. In the female, the upper arrow points to a visible faint sclerotization of sternite 8 and the lower arrow points to the fused gonocoxites. In the male, the arrow points to sternite 8. (Images originally published in Kosnicky 2019, Journal of Insect Science)

Once these methods were made, he then took the same types of measurements and photographs with living H. comalensis taken from the San Marcos refuge.

Of all the measurements taken, only the elytron length showed potential for distinguishing between sexes of S. comalensis. Individuals with elytrons longer than 2.06 millimeters were considered females, but this measurement still misidentified living females six out of 11 times and males three out of nine times.

Lateral lighting of preserved specimens and living beetles, however, could illuminate internal organs that proved to be better at distinguishing sexes. In general, lateral lighting could provide clear views of the abdominal organ sternite 8 in males and fused gonocoxites in females. The abdominal sternite 8 was either light or unperceivable in females. These differences were confirmed by dissection of preserved specimens, and similar abdominal features were revealed by lateral illumination of live H. comalensis insects.

Under a lateral lighting setup, the female (left) and male (right) of the Heterelmis comalensis, sometimes known as the Comal Springs riffle beetle, can be differentiated. In the female, the upper arrow points to the anterior strut of sternite 8 and lower arrow points to sternite 8. In the male, the arrow points to sternite 8. (Images originally published in Kosnicky 2019, Journal of Insect Science)

The study presented the most accurate method so far of determining sex among the endangered beetles. “It was rare to encounter an adult S. comalensis or H. comalensis that was not readily identifiable as a female or male using the characters and lighting techniques described [in the paper],” Kosnicki writes.

Photography was shown to play a key part in identifying males and females, but Kosnicki warned that photography still lacks the resolution seen in live viewing with a stereoscope. “As imaging systems are now becoming standard equipment in laboratories, there seems to be a shift to looking at specimens on a monitor rather than looking through eye pieces. Many fine details are lost on screen,” he says.

And these details, the study showed, could become important as breeding efforts are used to help preserve endangered insects like H. comalensis and S. comalensis.

Andrew Porterfield is a writer, editor, and communications consultant for academic institutions, companies, and nonprofits in the life sciences. He writes frequently about agriculture issues for the Genetic Literacy Project. He is based in Camarillo, California. Follow him on Twitter at @AMPorterfield or visit his Facebook page.