Exploring the temperature tolerance of a cave beetle

Methods

Summary

Field collections

The animals we work with these days stem from collections in 2012 and 2013 in two entrance areas of Mammoth cave as part of our onsite research activities covered by Scientific Research and Collecting Permit #MACA-2011-SCI-0001, which also enables future collections if needed.

Cave beetle laboratory culture

We are culturing P. hirtus following the detailed yet straightforward protocols developed by Stewart Peck (1975) with minor tweaks. The animals are kept in 150 mm polystyrene Petri dishes supplied with native cave soil. To reproduce the saturated humidity of the cave environment, the Petri dishes are sealed with an elastic but oxygen permeable parafilm and seated on moist tissue inside styrofoam boxes. The boxes are kept in a light-insulate cold chamber set at 11 degrees Celsius. To rule out light stress damage to the likely very sensitive P. hirtus eyes, the cave beetle cave box is situated in a completely light-insulated room. All inspections and handling of cultures are conducted under red light. Consistent with our genetic data (Friedrich et al. 2011), preliminary behavioral data indicate that the beetles are insensitive to red light (check out the phototaxis movies in our more detailed background presentation by clicking HERE). As is typical for cave animals, P. hirtus is used to subsisting on little energy input. The cultures are doing fine, being fed only every other week with a few granules of dry baker’s yeast. The slow reproduction and development of P. hirtus is thus compensated by low maintenance needs and costs.

Monitoring culture health

Each feeding, we count larvae, pupae, and adult animals per plate. Extending the culturing records by former Master’s student Jasmina Kulacic, who graduated in summer 2014, we have now over three years of data on larval and adult viability. This is a solid reference to compare future culturing success with.

Defining the short-term temperature tolerance range

To define the temperature tolerance range of P. hirtus in the pilot series of experiments, we will culture sample populations of 10 individuals at temperatures between 4-30 degrees Celsius [or higher pending results] in 2 degrees increments. For each temperature point, Sonya will be record the motoractivity of larvae and adults, survival of larvae and adults, and egg laying rate every two days for two weeks. The upper and lower bounds of short-term temperature tolerance will be defined as the temperature range with no detectable immediate negative impact on adult and larval survival and activity compared to standard culturing at 11 degrees Celsius.

Determining the long-term temperature tolerance range

To determine the temperature range for optimal culturing and explore the long-term temperature tolerance of P. hirtus, we will proceed to conducting experiments, in which we monitor the vitals of tester populations for up to six months at different temperatures within the short-term tolerance range. While time consuming, this approach is essential for capturing the long-term effects of temperature on reproductive success and developmental speed. Final decisions on the temperature range to be explored will be made based on the findings in our the short-term temperature tolerance study.

Challenges

All culture handling is performed in our light-insulated cave laboratory room aided with red light head lamps. One major challenge we anticipate related to defining the optimal culture temperature range is that the growth of microorganisms may be stimulated at higher temperatures which possible negative consequences on P. hirtus population health.