Uso del tiempo libre

Hemolymph volume was estimated gravimetrically because the inulin dilution method cannot be used, as the heart does not function in chill-coma. Instead, I corrected my estimates of hemolymph volume and accounted for residual hemolymph content of muscle samples (4.14 % w/v) using the inulin dilution technique (Ziegler et al., 2000; Qi et al., 2004) on 19 crickets at 22 °C temperature (Figure 5.1). A 5 % w/v solution of FITC inulin in 150 mM NaCl was dialyzed in the dark and at room temperature against 150 mM NaCl (to remove unbound FITC) in benzoylated dialysis tubing with a molecular weight cut-off of 2 kDa (Sigma Aldrich, St. Louis, MO, USA) and the resulting solution (~3 % w/v FITC-inulin) was used for injection.

Figure 5.1. The relationship between gravimetric estimates of hemolymph volume and volume quantified using the inulin dilution method in G. pennsylvanicus.

Gravimetric estimates of hemolymph volume significantly correlated (Pintercept < 0.001;

Pslope < 0.001, R2 = 0.66) with estimates obtained on the same individuals using the inulin dilution method.

Volume (gravimetric; µL)

100 120 140 160 180

Volume (Inulin dilution; µL)

0 50 100 150 200 250 80 60 40 20 0

Crickets were immobilized using plasticine and vacuum grease was applied to the injection site to minimize bleeding. A 2 µL bolus of the inulin solution was injected at the base of the right wing under the pronotum. Fifteen minutes later the crickets were sampled as above to collect hemolymph and muscle tissue. Muscle tissue from one femur was used to measure water content gravimetrically. The hemolymph and muscle tissue from the other femur were weighed and placed in 20 volumes of 500 mM HEPES buffer (pH 7.4), homogenized at 22 °C using a bullet blender with 1 mm glass beads (Next Advance, Averill Park, NY, USA), and centrifuged at 7000 × g for 10 minutes. 50 µL aliquots of the resulting supernatant were loaded onto black 96-well plates and fluorescence measured (excitation=485 nm, emission=538 nm) in a Spectramax M2e spectrophotometer (Molecular Devices, New Milton, Hampshire, UK). Sample fluorescence was compared to dilutions of the 3 % injection solution in 500 mM hepes and corrected by comparison to hemolymph and muscle tissue of three control crickets that did not receive an injection. Gravimetric estimates of hemolymph volume significantly correlated (Pintercept<0.001; Pslope<0.001, R2=0.66) with estimates obtained

on the same individuals using the inulin dilution method.

Crickets were cooled from 25 °C at 0.25 °C min-1 and held at 0 °C. After 24 h, ten crickets were removed to 4 °C and immediately dissected (t=0; ~3 min per dissection), while 40 crickets were removed to 22 °C and dissected after 5, 20, 60 and 120 min of recovery (n=10 at each time point). Control crickets were dissected directly from rearing conditions (25 °C). Tissue collection and ion content analysis followed previously described methods (Chapter 4). Briefly, hemolymph was collected from incisions at the base of each hind limb, the thorax and abdomen were then opened dorsally and any additional hemolymph was collected. Muscle tissue was collected from the hind femurs and blotted gently to remove residual hemolymph. Foregut, midgut and hindgut were ligated (to retain their contents) at the junctions between the proventriculus and midgut, and midgut and hindgut, before being removed. All tissue samples were placed into pre- weighed 200 µL tubes. Tissue water content was determined gravimetrically from the mass before and after being dried at 70 °C for 48 h. A 200 µL aliquot of nitric acid was added to the dried samples which were digested for 24 h at room temperature and stored at -20 °C for up to 3 weeks before analysis. Total tissue Na+ and K+ content were

determined using atomic absorption spectroscopy (iCE 3300, Thermo Scientific, Waltham, Ma, USA) by comparisons to standard curves. Muscle equilibrium potentials were calculated using the Nernst equation.

5.2.4

Respirometry

I used open-flow respirometry to measure mass-specific rates of CO2 production (VCO_!) as a proxy for metabolic rate during recovery from a range of exposures to 0 °C (1-24 h). CO2 production was recorded using flow-through respirometry (Chapter 3; Williams et al., 2010). Briefly, crickets were individually placed into a glass 11 cm3 chamber in a temperature-controlled cabinet (PTC1, Sable Systems International (SSI), Las Vegas, Nevada, USA). Activity in the chamber was detected with an AD-1 infrared activity detector and temperature by a 36 AWG type-T thermocouple held in place against the outside of the glass near the cricket. Dry, CO2-free air was pushed through the chamber at 50 mL·min-1 (mass flow valve: Sierra Instruments, Monterey, California, USA and control unit MFC2, SSI). [CO2] and [H2O vapor] were measured in the excurrent air (Li7000, LiCor, Lincoln, Nebraska, USA). All data were recorded via a UI2 interface and Expedata software (SSI), and baseline recordings made for 10 min before and after each run using an empty chamber. [CO2] was corrected for dilution by water vapor and converted to VCO! (mL min-1 g-1).

A total of 45 crickets were used to record VCO_! during chill-coma recovery. Prior to cold exposure, crickets were removed from their rearing conditions and held for 2 h in the respirometry chamber before VCO! was measured for 1 h at 22 °C. They were then

transferred to a 14 mL plastic tube, which was placed in a well in an aluminum block cooled by fluid circulating from a Proline RP855 circulating bath (Lauda, Wurzburg, Germany). The crickets were allowed 15 min equilibration at 22 °C before being cooled at 0.25 °C min-1 _{to 0 °C where they were held for 1 - 24 h. After cold exposure, each} cricket was placed upright in the respirometry chamber at 22 °C, and VCO! recorded

during recovery. The time of the first movement of the cricket was recorded from the activity trace. The duration of recording VCO_! during recovery was modified based on preliminary observations of metabolic overshoot: 4 h (1-2 h at 0 °C), 6 h (3-8 h at 0 °C)

or 8 h (9-24 h at 0 °C). Six control crickets that were not exposed to cold were recorded twice in the respirometer with a one hour rest at 22 °C between recordings. There was no difference in VCO_! between the first a second recording (P=0.102), and no evidence of a metabolic overshoot during the second recording.

5.2.5

Data analysis

Statistical analyses were conducted with R (v. 2.13; R Core Development Team, 2011). Linear and exponential models of the relationship between cold exposure duration and chill-coma recovery times were fitted using the gnls() function in R and compared with the Akaike information criterion (AIC), where ΔAIC ≥ 2 indicated a better model.

To account for changes in both ion and water content during cold exposure, ion data are expressed as both concentration and total tissue content. Tissue water and ion content both significantly correlated with tissue dry mass (P<0.001), so analyses of water and ion content of the tissues (excluding those of the hemolymph) were performed on residuals of a regression of content and dry mass. Ion concentrations, ion content and water content for each tissue were first compared between control conditions and immediately following cold exposure using t-tests followed by false discovery rate (FDR) correction (Benjamini and Hochberg, 1995) using the p.adjust() function in R. The relationship between time and water or ion content during chill-coma recovery were then determined by fitting to both linear and exponential models using the gnls() function in R, and models FDR corrected and compared with AIC as above.

Crickets were excluded from the respirometry analyses if they died within 24 h of the experiment (n=3) or displayed excessive activity during respirometry (n=7). Respirometry activity data were converted to absolute difference sum (ADS; Lighton and Turner, 2004). A ten-minute period with the lowest ADS slope, indicating minimal activity, was used to calculate a mean VCO_! for each cricket prior to cold exposure (Chapter 3). Mean VCO_!following cold exposure was estimated using the same method for only the final 25 % of the recording to ensure metabolic rate had stabilized post-cold exposure. Pre- and post-cold exposure VCO! data were compared using the aov() function (with mass as a covariate) to examine the effect of cold exposure on resting metabolic

rate. To estimate the time at which the transient rise in metabolic rate during chill-coma recovery was completed, the point at which metabolic rate crossed the mean metabolic rate post-recovery as determined above was used. The total time required for the metabolic overshoot to end was then determined from the point at which the cricket was removed from the cold and placed in the respirometry chamber at 22 °C.

The exponential loss of Na+ _{content from the hemolymph as a function of cold exposure}

duration was estimated using compiled Na+ _{content data from the present study and from}

a previous study of the same cricket population (Chapter 4). The time required to restore

Na+ content was then estimated using the slope of the linear regression of hemolymph

Na+ content against time (Figure 5.3C), and these rates were used to estimate the time

required to restore hemolymph Na+ content to control levels as a function of cold

exposure duration.

Detailed statistical results of model fit for this chapter are presented in Appendix B (Tables B.1-B.3).