DNA Analysis Reveals Parasite Diversity in Black Flies

Simulium sp. black fly — Flies in the order Simuliidae, known as “black flies,” such as this fly in the genus *Simulium*, can carry blood parasites that cause the malaria-like bird disease leucocytozoonosis. While the parasites can’t survive the human immune system to cause disease, bites from black flies can still be painful and cause unpleasant reactions in humans, such as fever, headache, nausea, and swollen lymph nodes. (Photo by John Rosenfeld, via BugGuide.net, republished with permission)

By Melissa Mayer

While some blood-feeders take a delicate approach to enjoying a meal, black flies emphatically do not: They use their jagged mandibles to tear into flesh and then slurp up the pooled blood. And sometimes they leave a parasite behind.

In a study published April 7 in the Journal of Medical Entomology, researchers at Mississippi State University looked for the presence and diversity of parasites from the order Haemosporidia in black flies collected in their state. Their results were positive for haemosporidian DNA, and a closer look at the DNA sequences revealed that some of the parasites recovered in the study, species in the genus Leucocytozoon, diverge from those already in the database.

Meet the Haemosporidians

Leucocytozoon blood parasites cause the malaria-like bird disease leucocytozoonosis and rely on flies from the order Simuliidae, known as “black flies,” to transmit them from one host to another. It’s a problem for both domestic and wild birds, who may show no signs of disease or experience mild to severe symptoms or even large die-offs. While the parasites can’t survive the human immune system to cause disease, bites from black flies can still be painful and cause unpleasant reactions in humans, such as fever, headache, nausea, and swollen lymph nodes.

Beginning in 2008, more and more reports of human bites and backyard poultry deaths began to trickle into the Mississippi Department of Health and the Mississippi State University Extension Service. Experts noted black fly outbreaks in the years 2011, 2012, and 2018.

“At the end of the day, it’s an economic problem,” says Jessica Ber, lead author on the study and now an environmental specialist at the Florida Department of Agriculture and Consumer Services.

“You want your poultry to be big and healthy and happy, and the black flies alone—how they feed and swarm—can cause stress in the poultry,” she says. “On top of that, you’ve got this disease that can cause anemia and just make them really sick and their eggs really small. So, overall, it can affect the entirety of the poultry farm.”

Ber says one of the problems with black flies is that they aren’t well known. “They call them gnats sometimes,” she says. “They get them mixed up with biting midges a lot because they’re both really teeny tiny. … So, some education out there about black flies would be good. But also knowing what’s out there is equally important. We need to know what’s potentially infecting our poultry.”

Sequencing and Phylogenetics

Ber’s study, which she worked on as a side project during her master’s program, was born out of circumstance. Her advisors and coauthors Jerome Goddard, Ph.D., and Tina Nations, Ph.D., collected black flies for another purpose and suggested running the data to see what parasites they were carrying. Once the insects were confirmed to be black flies, Ber minced them with sterile razors and extracted DNA. She used a technique called polymerase chain reaction (PCR) to amplify the haemosporidian DNA before sending it out for sequencing.

Then Ber ran the sequences against the National Center for Biotechnology Information (NCBI) BLAST database, which helps infer relationships among sequences. It turned out that 21 of the 186 pooled samples contained haemosporidian DNA, and 18 of those were Leucocytozoon species. Even more interesting, nine of those Leucocytozoon species’ DNA sequences differed from the sequences already in the database. Ber’s advisor and coauthor Diana Outlaw, Ph.D., specializes in phylogenetics—which looks at how species or sequences are evolutionarily related to each other—so she drew up a phylogenetic tree and confirmed that some of the Leucocytozoon lineages group together but don’t seem closely related to any known lineages.

That moment was an exciting one. “Seeing that tree for the first time, even though I’m not a phylogeneticist by any means, and I definitely had to have some explanation … and seeing how they were separating, that was probably the coolest moment,” Ber says.

Leucocytozoon cladogram — Analysis of haemosporidian DNA from collected black flies in Mississippi found nine *Leucocytozoon* species’ DNA sequences that differed those previously known. A phylogenetic analysis—which looks at how species or sequences are evolutionarily related to each other—shown in this phylogenetic tree (cladogram) confirmed that some of the *Leucocytozoon* lineages group together but don’t seem closely related to any known lineages. (Image originally published in Ber et al 2022, *Journal of Medical Entomology*)

More to Learn

What that means is still an open question. The lineages could be undescribed species—or it could mean the DNA simply wasn’t in the database yet. It suggests that there could be more Leucocytozoon diversity out there than previously recognized.

“Really, what it means at the end of the day is that more work needs to be done,” says Ber. “And that more surveillance needs to be done. We need to really see what is out there in these black flies, which are, compared to mosquitoes, more understudied.”

The team hopes that future studies—or perhaps studies already underway—will help shed more light on black flies and the parasites they carry.