Does Thermal Variability Experienced at the Egg Stage Influence Life History Traits across Life Cycle Stages in a Small Invertebrate?

Although effects of thermal stability on eggs have often been considered in vertebrates, there is little data thermal stability in insect eggs even though these eggs are often exposed in nature to widely fluctuating ambient conditions. The modularity of development in invertebrates might lead to compensation across life cycle stages but this remains to be tested particularly within the context of realistic temperature fluctuations encountered in nature. We simulated natural temperate fluctuations on eggs of the worldwide cruciferous insect pest, the diamondback moth (DBM), Plutella xylostella (L.), while maintaining the same mean temperature (25°C±0°C, 25±4°C, 25±6°C, 25±8°C, 25±10°C, 25±12°C) and assessed egg development, survival and life history traits across developmental stages. Moderate fluctuations (25±4°C, 25±6°C) did not influence performance compared to the constant temperature treatment, and none of the treatments influenced egg survival. However the wide fluctuating temperatures (25±10°C, 25±12°C) slowed development time and led to an increase in pre-pupal mass, although these changes did not translate into any effects on longevity or fecundity at the adult stage. These findings indicate that environmental effects can extend across developmental stages despite the modularity of moth development but also highlight that there are few fitness consequences of the most variable thermal conditions likely to be experienced by Plutella xylostella.


Introduction
Because natural diurnal fluctuations in temperature can impose different impacts on ectotherms when compared to constant temperatures [1], it is important to assess the impact of thermal stability on ectotherm performance [2]. In organisms with complex life histories, temperature fluctuations at one life cycle stage might affect phenotypes at other stages [3]. Fluctuations are likely to be particularly important at the egg stage because this stage is frequently exposed to diurnal fluctuating temperatures in nature [4] and cannot escape thermal extremes in the same way that larvae and adults can behaviorally thermoregulate.
A number of studies have explored the effects of egg temperatures on life history performances in vertebrate ectotherms [5] and these studies have shown that egg temperature impacts egg traits [6] and also post-hatching traits [7]. On the other hand, invertebrates are different from vertebrate ectotherms by exhibiting a modularity of life cycles that may assist in dealing with environmental variability by possible insulation of later stages from disturbances during embryogenesis [8]. The egg stage of invertebrates is thought to be susceptible to thermal stability because of rapid rates of heat transfer and short durations [9] that make them sensitive to thermal fluctuations involving brief high temperature exposure [10] and recovery normal temperature intervals [11].
Short term stress exposures have been used to explore the thermal stress effects on the egg stage [12], although these types of studies do not consider the effects of daily fluctuations that might lead to cumulative effects through injury when stressful low night temperatures are followed by stressful daytime temperatures [13,14]. In some relevant studies, changes to temperature regimens involved both means and fluctuating ranges simultaneously [15], making it hard to separate out the effects of fluctuations per se. Moreover the extent to which fluctuating conditions at the egg stage influence later stages is largely unexplored.
The diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae), is the most destructive pest of cruciferous vegetables, and is widely distributed from tropical to cool temperate regions where substantial fluctuations in temperature are experienced [16,17]. In this species it is well known that changes in constant temperatures affect life history traits such as growth, development, survival, reproduction [18] and migration [19].
Here we consider the following questions: (1) Do thermal fluctuations of the magnitude experienced in nature during egg stage affect egg hatch in DBM? (2) Do any fluctuations experienced at the egg stage have subsequent effects at the larval, pupal or even adult stage? We incubated eggs of DBM at a constant temperature of 25uC and five fluctuating temperature of 2564uC, 2566uC, 2568uC, 25610uC and 25612uC and considered effects on development time and survival of eggs, larval and pupal development time, larval growth rate and survival, adult longevity and fecundity.

Insect rearing
The population of DBM larvae was collected from Brassica fields in May 2010, with the permission of the Experiment Station of Hubei Academy of Agricultural Sciences, in Wuhan, Hubei Province, China. All stages of DBM were reared in an insect rearing room with artifical diets (Southland Products Incorporated, USA) at a constant 25uC, 50%-70% RH and a photoperiod of L16:D8. DBM had been reared under these conditions for at least 10 generations before this study. After pupation, 200 pupae (1:1 sex ratio) [20] were transferred to a screen cage (35620635 cm) for adult emergence. Once adults emerged, a cotton ball immersed in 10% honey water solution was supplied for adult feeding. 93 female adults eclosed and laid eggs. Two pieces of laboratory film (765 cm) were immersed in fresh juice of cabbage leaves for 3-5 seconds and then hung from the top of the screen cage at 15:00-16:00 h for egg laying. The film laid with DBM eggs was taken out at 7:00-8:00 am the next day. About 300 eggs on the film were counted and put on the surface of artificial diets (120 g) in a plastic rearing box (1061069 cm). The rearing box was kept at the rearing room until pupation and then followed the same procedures that were described above for pupae and adults rearing.
Design and manipulation of fluctuating temperature cycles A 24-h temperature cycle was used to simulate temperature fluctuations in a cabbage field in summer. Daytime temperature lasted for 8 hours (08:00-16:00) with the daily maximum temperature (DTmax) at 14:00, and the night temperature lasted for 16 hours (16:00-08:00) with the daily minimum temperature (DTmin) at 4:00. Temperature changed gradually between DTmax and DTmin. One constant temperature (25uC) and five fluctuating temperatures were used: 2564uC, 2566uC, 2568uC, 25610uC and 25612uC. We selected 25612uC as the widest diurnal range, because the temperatures in the microhabitat which DBM would experience in cabbage fields fluctuated daily within this range in summer in Beijing (Fig. 1). In addition, the mean temperature experienced in these fields is around 25uC.
Temperatures in different climate chambers were logged every 20 minutes (U23-001, Hobo Ltd., USA) and showed that average temperatures in the chambers were around 25uC (Table1, Fig. 2 A-B). Relative humidity in chambers was 50%-70% and photoperiod was set to 16:8 (L:D) during the experiment.

Growth, survival and reproduction
Plates were sterilized for 30 min by ultraviolet light, and each well was filled with 1.7 ml prepared artificial diet for DBM feeding. Three new eggs (laid within the last 12 h by 1-2 days old females) were transferred into each well with a fine camel's hair brush. To facilitate ventilation, the plates were covered with fine nylon mesh. Three plates were placed in each climate chamber. Over 200 (3 eggs*24 wells*3 plates = 216) eggs were observed in each temperature regimen. Hatching status of eggs was checked (with a stereo microscope) twice daily at 08:00 and 20:00, because temperatures in climate chambers at these times were similar to room temperature (about 25uC).
After eggs hatched, all tested insects were moved to a 25uC rearing room. Newly hatched larvae from different treatments were randomly assigned to wells (1 larva per well). Ninety six larvae (24 wells *3 plates = 72) were used for each temperature treatment. The pupation status was checked daily at 08:00 until all larvae had died or developed to pupae. When larvae developed into pre-pupa [21], they were weighed. Plates were renewed every  three days to keep artificial diets fresh. When larvae became spindle-shaped and had a thin white silk cocoon, they were recorded as having entered the pupa stage [22]. Pupae continued to be observed at 08:00 am until all adults emerged or pupae died (based on lack of adult emergence). Survival and development time were recorded for all immature stages. Newly emerged male and female adults which were from the same replicate under the same egg temperature treatment were paired and transferred into a glass tube (3612 cm) for mating and egg-laying. Two sides of the tube were covered with the fine stainless steel mesh for ventilation. A piece of cotton immersed with honey water solution (10%) was placed in the tubes for adult feeding. At 15:00-16:00, a piece of laboratory film was inserted to the tube for egg laying. The adults were transferred into a new tube with new film at 7:00-8:00 every day until females died. All eggs laid on the film and inner surface of the tube were counted.

Statistical analysis
Effects of different temperature treatments during egg stage on all indices of growth, survival and reproduction were analyzed with ANOVA in which egg temperatures and adult sex were treated as fixed factors, and replicate plates as a random factor (nested within egg temperatures). Means were compared using Duncan's multiple range test in SAS V8 (SAS Institute, Cary, NC). We analyzed effects of different temperature treatments during the egg stage on development time of eggs/larvae/pupae, growth rate of larvae, percent hatching rate (number of larvae/number of eggs6100), percent pupation rate (number of pupae/number of larvae6100), percent emergence rate (number of adults/number of pupae6100), adult fecundity and longevity. Growth rate of larvae were expressed as mg/d (pre-pupal mass/development duration of larvae).

Effects of thermal fluctuations on eggs
Temperature fluctuations affected development time of the egg stage, but only at high fluctuations of 610uC. This suggests that a temperature range of 17-33uC may not have much impact on egg development time if the mean temperature is 25uC. The impact of thermal stability on egg development is thought to be related to the upper and lower thermal limit for egg development [23]. Within an optimum range, developmental responses to temperature are expected to be approximately linear [24]. Daytime temperatures higher than the mean temperature and thus accelerate egg development, but the nighttime temperatures lower than the mean and slow development. With regular sinusoidal fluctuating temperatures as in our experiments, a uniform rate of development of DBM eggs is expected under moderate fluctuations in temperature, in contrast to fluctuations encompassing extreme values that depress development [25]. According to Jensen's inequality and the Sharpe-DeMichele model, development should slow if daytime temperature falls over the upper threshold or nighttime temperature falls below the lower threshold for development [26]. The maximum temperature of 37uC in our experiment is higher than 34uC, the upper threshold for development of DBM [27], for 6 hours (0.25 day) per day. Such high temperatures not only fail to compensate slower development at lower nighttime temperatures (,20uC), but may also depress development [27]. If we assume that there is no development above the upper threshold, we can largely account for the slower egg development at wide thermal fluctuations. Development time at 25612uC, 3.86d is very close to the sum (3. In contrast to effects on development time, temperature fluctuations had little impact on egg survival. Previous work has shown that egg survival is impacted by high temperature [28] and stress exposure time [29]. Hatching rates of DBM eggs are not changed much within a constant 8-31uC temperature range, but Table 2. Results of ANOVA testing the effects of egg temperature treatments, sex, and replicate plates (as a random factor, nested within egg temperatures) on development time of egg, larva or pupa, pre-pupal mass, larval growth rates, adult longevity, female fecundity, hatching rate, pupation rate and emergence rate in Plutella xylostella (L.). decreased to 58% at 32uC [30] or a slightly higher temperature [17]. However, in our results DBM eggs survived well even when temperature fluctuated as widely as 612uC in which the daily maximum reached 37uC, far over the constant upper lethal 34uC limit. This high survival may relate to recovery from heat injury during optimum nighttime temperatures [31] or inadequate damage to affect survival. Similar patterns of recovery under fluctuating temperatures have been found in other species [32,33]. Wide fluctuations of temperature may improve survival under heat stress but delay egg development [34]. Under a diurnal fluctuation  cycle, daytime high temperatures induce the rapid synthesis of protective factors such as hsp70, mannitol and sorbitol, increasing heat resistance [35,36], while nighttime temperatures reduce metabolism and energy consumption [37] and provide optimal thermal conditions for heat injury recovery.

Effects of egg treatments across developmental stages
Our results indicate that egg thermal stability significantly impacted the growth of larvae. Body mass and growth rate increased significantly in larvae hatched from eggs which experienced wide fluctuations. Compensatory growth can occur if organisms suffer from nutritional stress [38] or temperature stress [39] during early development. Wide temperature fluctuations in the egg stage may be stressful and trigger compensatory growth to increase larval development time. Stress during organisms' early stage does not change the number of cells [40], but results affect energy metabolism and utilization [41]. Stress in the egg stage often induces larval compensatory growth through accelerating food intake or increasing intake time (the latter extending development) [42]. Our results indicate development time was not extended (Fig. 4A, 5A), but body mass was increased (Fig. 4C) perhaps reflecting an increase in food intake. Compensatory growth during the larval stage may therefore be a life history strategy in DBM to buffer the impact of variable thermal conditions during the egg stage.
Thermal stability did not change other traits in larva, pupa or at the adult stage. The lack of long term fitness effects may reflect successful compensation and/or the fact that these insects undergo complete metamorphosis, such that different stages experience a physiological and morphological body rebuild during metamorphosis, creating a high degree of independence for each stage [8]. This may also help explain why early stage stress in complete metamorphosis insects does not affect adult fluctuating asymmetry [43], whereas such effects are detected in species without complete metamorphosis [44].

Pest management implications
Survival and phenology are important components of population models for monitoring, forecasting, control and harvesting in resource or pest management. Because the performance of organisms under [45] constant temperature regimens are well known in many species [46,47], it is important to assess thermal conditions under which data from constant temperature experiments can be applied. Our results indicate that the traditional linear model for ''constant temperature-development'' can be widely applied if the mean temperature is optimal and the daily fluctuation is not very wide such as a mean of 25uC and fluctuation less than 68uC for DBM [45]. However, these models may not describe development in an area or period with low or high mean temperature and/or wide diurnal temperature range. Survival data obtained at constant temperatures appear applicable to a wider range of temperatures. For practical applications, we suggest that when temperatures range between 17-33uC at the egg stage, the results from constant temperature experiments are applicable, but the diurnal temperature range should be incorporated into the fecundity in the egg of DBM following emergence from eggs exposed to different fluctuating temperature ranges (2560uC, 2564uC, 2566uC, 2568uC, 25610uCand 25612uC). doi:10.1371/journal.pone.0099500.g005 development model and perhaps survival model when temperatures are known to fluctuate widely [48].
Recently, trends and future climate scenarios suggest a reduction in diurnal temperature ranges across the world [49]. This indicates that diurnal temperature range, as an independent factor, has potential impacts on population dynamics of DBM. In low latitude areas such as southern China [50] and southwestern Europe [51], the diurnal temperature range is below 610uC at which the development of DBM can hardly be influenced and is similar to that at constant. However, in high latitude areas such as northwest China and central Russia (http://cdc.cma.gov.cn/ home.do), the diurnal temperature range is above 610uC at which the development of DBM may be restricted and is slower than at constant temperature. With diurnal temperature range decreasing in high latitude areas in the future, we presume that the development of DBM could be accelerated and consequently the number of generations could be increased. In this case, the potential risk of DBM will be increase during the growing season of Brassica crops.