So, researchers at Texas A&M University decided to dig deeper—literally. Over a 17-month period, they dug up leaf litter and soil samples in an area known to be well-populated with adult blacklegged ticks (Ixodes scapularis) and searched for larvae and nymphs there. Their hypothesis was that the young ticks descend into the ground layers to seek moisture and evade the dry Texas heat.

Dragging a cloth on the ground is a standard method for collecting ticks in their habitat. Paired with sweeping the cloth across low vegetation, the method is known as “flagging and dragging.” In the northern United States, flagging and dragging regularly collects blacklegged ticks (Ixodes scapularis) of all life stages: larva, nymph, and adult. In the southern U.S., though, there’s a peculiar difference: The method only turns up adults. (Photo credit: Mackenzie Tietjen)

“We definitely expected to find them in the leaf litter or at least the soil underneath the leaf litter. We thought we would be able to use this method to document their phenology in Texas,” says Mackenzie Tietjen, a Ph.D. student in the Department of Entomology at Texas A&M who led the study.

But, 600 soil samples later, not a single blacklegged tick had been found. Zero. None.

Tietjen and her advisors Maria Esteve-Gassent, Ph.D., and Raul F. Medina, Ph.D., published the results of the study this month in the Journal of Medical Entomology.

Blacklegged ticks are the primary vectors for the bacteria, Borrelia burgdorferi, that causes Lyme disease. In the northeastern U.S., where Lyme disease prevalence is highest, nymph-stage ticks play a key role in Lyme disease epidemiology because their small size—just 1 millimeter long—makes their bites more likely to go undetected long enough to transmit the Lyme disease pathogen. The nymphs’ absence from vegetation or even leaf litter and soil in the southern U.S., however, may be related to the lower prevalence of the disease there.

“Understanding the ecology of Lyme disease is one of the best tools for developing sustainable and effective disease control methods,” says Tietjen.

Blacklegged ticks are indeed present in the southern U.S. All stages—larvae, nymphs, and adults—are regularly found on their animal hosts. And, in fact, Tietjen and colleagues found more than 600 adult blacklegged ticks loose in the environment via flagging and dragging near the same test site. But the question remains: If immature blacklegged ticks not attached to their hosts can’t be found on vegetation or in leaf or soil layers, where are they? In her report, Tietjen proposes a working hypothesis.

“Because these ticks are prone to desiccation and it is so hot in Texas, they must be in a protected environment. So, I was thinking about other tick’s life histories that are nidicolous—i.e., living in their hosts’ nests—and wondered about the possibility of a stage-specific, mixed host-seeking strategy, and proposed the hypothesis that perhaps in the south blacklegged tick nymphs and larvae are found in their vertebrate host nests” she says.

Other tick species are known to live only inside their hosts’ nests rather than actively seeking out hosts above ground, and at least a few are known to employ both strategies at different life stages. Tietjen speculates that blacklegged ticks in hotter climates may have adapted to do the same, living in hosts’ nests when young but seeking other hosts above ground as adults. That would mean the next place to look would be lizard burrows, as lizards common in southern states are the primary hosts for immature blacklegged ticks, rather than small mammals, as in northern areas.

To test soil and leaf-litter samples for the presence of immature blacklegged ticks, researchers at Texas A&M University used Berlese funnels. In collection efforts using this method, the samples are placed in the funnels under heat lamps, and any arthropods in the samples migrate downward to avoid the heat and desiccation caused by the lamp. At the bottom of the funnel, the arthropods are collected in a jar. (Photo credit: Mackenzie Tietjen)

Tietjen says she and her colleagues hope to conduct such a study in the near future. For now, though, she says it’s important to share their progress in this process of elimination.

“We are big advocates for publishing negative data, and this article is a great example of how, even though we didn’t find what we were expecting to, it still is really important for the research community to know we did not find ticks where several people thought they could be,” she says. “Not finding I. scapularis beneath the leaf litter in Texas has made us generate a new hypothesis about their whereabouts that we are eager to test. And we hope that other research teams try replicating this study to see if sampling leaf litter in other southern states returns the same results.”