Populations of Plectus murrayi, a mesophilic nematode, have been increasing in the McMurdo Dry Valleys, Antarctica over the past three decades. In contrast, most other Antarctic nematode species, including Scottnema lindsayae, have experienced constant population declines over the same time period due to climate change related increases in summer temperatures and wetting occurrences. To determine why P. murrayi populations are outperforming other Antarctic nematode species, we performed a laboratory experiment to measure their metabolic responses to increasing temperatures. For this experiment, P. murrayi were cultured from soils collected in Taylor Valley during the austral summers of 2016 and 2017 as part of the McMurdo Dry Valleys Long Term Ecological Research (LTER) Project. Using a TAM IV Isothermal Microcalorimeter, we measured the metabolic heat rates of these cultured P. murrayi populations in six sealed 4 mL vials. Three of these vials also contained 200 µl ampoules of 0.4 M NaOH. NaOH reacts with CO2 produced from metabolic processes to release a known amount of heat (108.5 kJ/mol CO2). The difference in metabolic heat rate produced in vials containing NaOH and those without NaOH is proportional to CO2 produced from P. murrayi metabolism. Consequently, this method allowed us to track metabolic heat rates as well as CO2 production rates. O2 consumption was calculated from vials without NaOH using Thornton's rule (455 kJ of metabolic heat/mol O2). P. murrayi metabolic heat rates were compared to C. elegans for reference, and baseline measurements included heat rates of E. coli and blank agar.
Nematode IsolationSoil samples were collected from Taylor Valley, Antarctica. Soil cores to 10 cm depth were removed using clean plastic scoops, placed in sterile Whirlpak® bags, and transported in insulated coolers via helicopter to McMurdo Station. The soil samples were gradually cooled to -20°C (rate of -10°C per 48 hours) and shipped frozen to Brigham Young University. Soils were then gradually warmed to +4°C (rate of +10°C per 48 hours). Nematodes were extracted from the soil using sugar density gradient centrifugation modified for Antarctic soils (Freckman and Virginia, 1997). P. murrayi were isolated and cultures established according to Adhikari et al (2010). Cultured Plectus murrayi were then placed in deionized water and stored at -20°C.
Agar liquid media was then prepared with double deionized water at a concentration of 15 g/L. Agar powder was stirred into the solution until homogenous translucent liquid formed and pH was adjusted to 7. The liquid media was autoclaved with a 20-minute sterilization step at 120°C and then poured into 60 mm petri dishes until they were approximately 2/3 full. Before agar was allowed to set, 2 g of sterilized Ottawa builder’s sand was added to the center of each plate. Sealed plates were held at room temperature for 2 weeks to monitor contamination.
Uncontaminated plates were then prepared with 40 µL of pure OP50 Escherichia coli culture that had been tested for contaminants and incubated at 37°C for 3 days. P. murrayi isolates which had been stored at -20°C were then thawed and deposited on E. coli plates and held at 11°C for a 4-week population expansion period. The living cultures were maintained by preparing additional agar plates with E. coli and using a sterile knife to transfer pieces of agar containing live nematodes from the old plates to the new ones. Agar transfers were carried out every 4 weeks to maintain the viability and health of the worms and to provide them with fresh E. coli.
Microcalorimetric Measurements of Heat and CO2 Production RatesA TAM IV isothermal microcalorimeter (TA Instruments, Lindon, UT) was used to measure metabolic heat and CO2 rates. Pieces of agar populated with a counted number of worms from a 2-week-old living culture of P. murrayi isolates were placed in each of six 4-mL vials with a sterile knife. A 250 μL flask was then added to each of the six vials: a flask containing 200 µL of 0.4 M NaOH in three vials, a flask with 200 µL of 0.4 M NaCl in two vials, and a flask with 200 µL of ddH2O in one vial (Criddle et al., 1991). The six 4-mL vials were then sealed and inserted into the six-channel calorimeter in the TAM IV.
During the experiment, CO2 produced by metabolism reacts with the NaOH to produce sodium carbonate and water, releasing 108.5 kJ of heat per mole of CO2. The difference in measured heat rate between the vials with NaOH and the vials without NaOH divided by 108.5 thus provides the rate of CO2 production (Criddle et al., 1990; Acar et al., 2004). O2 consumption was calculated from the measured heat rates from the vials without NaOH using Thornton’s Rule: 455 kJ of metabolic heat per mole O2 consumed.
The TAM IV was programmed to measure heat produced per vial at each of the following temperatures sequentially: 15°C, 10°C, 5°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 15°C. The 5-degree transitions between temperatures took approximately 1.5 hours. Vials were held at each temperature for 4 hours during which time heat rate measurements were recorded every 5 seconds. After a thermal equilibration period of about 30 minutes at each temperature, the measurements were averaged for each vial at each temperature setting. This experiment was then repeated using C. elegans without any NaOH for comparison. Baseline values for the heat rate measurements were obtained with a vial containing only agar, 200 µL of 0.4 M NaOH, and OP50 E. coli as well as a vial containing blank agar and 200 µL of 0.4 M NaOH.
The average heat rate for each vial with P. murrayi was then divided by the number of nematodes in the respective vial to calculate the average heat produced per nematode at each temperature. These measurements were then normalized to 30 nematodes and compared to the normalized C. elegans and baseline measurements. The average heat produced per P. murrayi individual at each temperature was then used to calculate the average rate of O2 production per nematode. The averaged heat rate per P. murrayi nematode in the vials without NaOH was then subtracted from the averaged heat rate per nematode from the vials with NaOH to obtain the average heat rate from CO2 reacting with NaOH at each temperature. This value was then used to calculate the average CO2 production rate per nematode at each temperature. The moles of CO2 produced per second per nematode was then divided by the moles of O2 consumed per second per nematode to calculate the respiratory quotient of P. murrayi metabolism.