Summary
According to the maternal manipulation hypothesis, females manipulate the phenotypes of their offspring by selecting favourable incubation conditions. In oviparous black ratsnakes (Elaphe obsoleta Say in James, 1823), females manipulate offspring phenotype through nest-site selection. This study aimed to determine whether the thermal mean and variance of the incubation regime affect fitness-related traits in hatchlings. We incubated 136 eggs in a split-clutch design at two thermal means (26 and 29 °C) and variances (constant and ±3 °C). Hatchlings incubated at higher temperatures hatched earlier, were longer, faster, and less defensive. Hatchlings incubated at constant temperatures hatched earlier and were longer. For athletic performance, there was a significant interaction between temperature mean and variance: hatchlings incubated at 29 °C swam faster, had a lower muscular strength, and righted themselves equally fast when incubated at constant temperatures, whereas hatchlings incubated at 26 °C were stronger, swam faster, and righted themselves more slowly. Overall, constant incubation temperatures produced hatchlings with phenotypes favouring higher survival than fluctuating temperatures, but the effect of thermal variance was not as pronounced as the effect of thermal mean. Therefore, we found some support for the hypothesis that black ratsnakes prefer communal over single-female nests because communal nests have higher, more constant temperatures.
Methodology
Study site
We studied ratsnakes in a 10 km × 3 km area surrounding the Queen’s University Biological Station, approximately 100 km south of Ottawa (Ontario, Canada). In this area, ratsnakes nest in large piles of decomposing vegetation that are loose enough to allow the snakes to burrow to depths of up to 75 cm (Blouin-Demers et al. 2004). Nests include large leaf piles, compost piles, and hollow tree stumps.
Egg collection and incubation
In July 2006, we collected eight clutches of eggs (n = 100) from known communal nests. Clutches are generally laid far enough apart in communal nests that they are easily distinguishable. We obtained an additional three clutches of eggs (n = 36) from gravid females caught opportunistically in the field and that subsequently laid in the laboratory. We equally and randomly divided each clutch among four treatments (n = 34 eggs in each treatment): two constant-temperature treatments (26 and 29 °C) and two treatments with means of 26 and 29 °C but fluctuating on a diurnal cycle (±3 °C). These temperatures were chosen to approximate those in communal and single-female nests: the diurnal fluctuations in single-female nests are approximately ±3 °C (Fig. 1b in Blouin-Demers et al. 2004), whereas communal nests do not fluctuate diurnally (Fig. 1a in Blouin-Demers et al. 2004). This split-clutch design was used to control for genetic and maternal factors that affect offspring phenotype. We placed the eggs in plastic containers so that they were half-buried in moist vermiculite (1:2 ratio by mass of vermiculite and water). The constant-temperature incubators were constructed from plastic containers insulated with closed-cell foam boards. Temperature was controlled with thermostats and heat was provided by 100 W light bulbs. Small fans ran continuously in each incubator to prevent any thermal gradients from forming. The fluctuating-temperature incubators were commercially made (Model LBC700, Constant Temperature Control Limited, Aurora, Ontario). We placed miniature temperature dataloggers (iButtons, Dallas Semiconductors Inc., Dallas, Texas) in the incubators to monitor the temperature. The mean temperatures (±95% CI) over the incubation period were 25.7 ± 0.035 and 29.5 ± 0.035 °C for the constant-temperature incubators, and 26.0 and 29.0 °C (precisely) for the fluctuating-temperature incubators (Fig. 1). Every 2nd day, we added water to each egg container to return it to its initial mass, which compensated for water loss via evaporation. We also shuffled the containers when replacing them in the incubators to compensate for any possible positional effects. Beginning in mid-August, we checked the eggs daily for signs of hatching, removed hatched neonates, and placed them in individually labelled containers.
Morphological and behavioural measurements
Morphological and behavioural measurements were made within 24 h of hatching. First, we assessed hatchlings for defensive behaviour upon first handling. We scored each individual on a scale from 1 to 5 (1, fleeing; 2, rattling tail; 3, gaping; 4, striking; 5, biting). Sex was assessed by eversion of the hemipenes. We measured snout–vent length (SVL) and tail length (TL) to the nearest millimetre using a ruler taped to the table and used the mean of two measurements in our analyses (Blouin-Demers 2003). We measured mass to the nearest 0.1 g on an electronic scale.