Effects of Temperature and Photoperiod on the Immature Development in Cassida rubiginosa Müll. and C. stigmatica Sffr. (Coleoptera: Chrysomelidae)

Tortoise beetles (Cassida and related genera) are a large cosmopolitan group that includes several pests of agricultural crops and natural enemies of weeds but their biology and ecology remain poorly known. Using a set of environmental chambers, we address simultaneous effects of temperature and photoperiod on immature development and adult body mass in two European species, C. rubiginosa and C. stigmatica. Consistent with its broader distribution range, the former species is less susceptible to low rearing temperatures, develops faster and has a larger body mass than the latter. However, C. rubiginosa seems to be less adapted to late-season conditions as a short-day photoperiod accelerates its immature development to a lesser extent than it does in C. stigmatica, which nevertheless results in greater larval mortality and slightly but significantly smaller adults. By contrast, in C. stigmatica, which is more likely to encounter late-season conditions due to its slower life cycle, short-day acceleration of development is achieved at no cost to survivorship and final body mass. The experiment with C. stigmatica was repeated during two consecutive years with different methods and the main results proved to be well reproducible. In addition, laboratory results for C. rubiginosa agree with field data from literature.

Temperature and photoperiod significantly influenced C. stigmatica survivorship (Table 1). There was a tendency for survival rates to be lower in cooler treatments and under short-day conditions (Fig. 1d-f), although there were quite a few exceptions to the latter pattern. For example, in the 2017 experiment, larvae actually survived better under short-day conditions (Fig. 1e), which is reflected in a significant photoperiod by year interaction in GLMs (likelihood ratio test χ 2 = 19.2, P = 0.00001). Maintenance of C. stigmatica larvae on living plants instead of cut leaves during the second experimental year significantly improved larval and, to a lesser extent, pupal survival (Fig. 1e,f, Table 1). In larvae, this ameliorating effect was observed at all but the lowest temperature of 16 °C, hence a significant temperature by year interaction (χ 2 = 5.0, P = 0.03).
Interaction terms in GLMs were generally non-significant with P-values of 0.5 and higher, but, when these interaction terms were included in the models, main effects tended to become non-significant as well. The model for C. stigmatica larvae mentioned above was the only exception where inclusion of double interactions did not affect the output; however, even in that case, addition of a triple temperature × photoperiod × year interaction  www.nature.com/scientificreports www.nature.com/scientificreports/ (χ 2 = 0.3, P = 0.6) rendered all of the remaining effects non-significant (χ 2 < 0.5, P > 0.5). Therefore, only the summaries of main-effects models are provided in Table 1.
The adult sex ratio in both species varied among the experimental regimens (Supplementary Tables S1 and S2) but this variation was not associated with rearing conditions (  Supplementary Fig. S1). There was a marginally significant effect of photoperiod, according to GLS models (Table 4), but the absolute differences between photoperiodic regimens were negligibly small, except at 16 °C, and inconsistent (Tables 2 and 3). Therefore, egg development data were pooled between photoperiodic regimens. There was no significant difference in egg developmental rates between C. rubiginosa and C. stigmatica, according to a GLS ANOVA with species and temperature as the only two predictor variables (effect of species: F 1,1629 = 0.2, P = 0.7). Lower temperature thresholds for egg development in these two species coincided and the sum of degree-days was almost identical (Tables 5 and 6).

Larval development.
Larval developmental rates showed a linear relationship with temperature in the studied range (Fig. 2b,e,f; Tables 5 and 6; Supplementary Fig. S1). Apart from the strongly pronounced effect of temperature, larval development in C. rubiginosa was significantly influenced by photoperiod and sex (Table 4). In particular, larvae developed slightly faster under short-day conditions at the two lower temperatures than in the corresponding long-day regimens (Table 2). However, despite the seemingly different response to photoperiod at different temperatures, there was no significant temperature by photoperiod interaction, which meant that the slope of the developmental rate-temperature relationship was unaffected by photoperiod (Fig. 2b, Table 5).
In C. stigmatica, larval development was significantly influenced by temperature, photoperiod, experimental year, and some interactions of these predictors (Table 4). Larval development was faster and the slope of the developmental rate-temperature relationship was steeper under short-day conditions relative to long-day conditions ( Fig. 2e,f; Table 6), hence a significant photoperiod by temperature interaction (Table 4). Rearing on living plants during the 2018 experiment resulted in significantly slower development, shallower slopes of thermal reaction norms, and enhanced effect of photoperiod relative to the previous year's results (Fig. 2e,f; Table 6), which was reflected in a significant triple interaction (Table 4).
In both species, larval males tended to develop more rapidly than females (Supplementary Tables S1 and S2), although only in C. rubiginosa this effect appeared significant (Table 4). There were no significant interaction www.nature.com/scientificreports www.nature.com/scientificreports/ terms including sex as a factor in the GLS models (P ≥ 0.001, Table 4), which meant that both sexes responded to temperature, photoperiod, and rearing method in a similar fashion, and so the developmental data in Tables 2, 3, 5, and 6 were pooled between sexes for the sake of brevity.
Lower temperature thresholds for larval development were similar in the two species and varied from 10.7 to 11.9 °C (Tables 5 and 6), but C. rubiginosa required a notably smaller sum of degree-days than C. stigmatica. In other words, larval development in the former species was more temperature-sensitive (the slopes of the thermal reaction norms were steeper) and uniformly faster than in the latter. This conclusion was well supported by a GLS ANOVA with species and temperature as predictor variables (2017 experiment, effect of species: F 1,975 = 1613.3, P < 0.00001; temperature × species: F 1,975 = 429.6, P < 0.00001).
Pupal development. As with eggs, pupal developmental rates strongly depended on temperature and were similar in C. rubiginosa and C. stigmatica, but the former species tended to develop slightly faster (Fig. 2c, Supplementary Fig. S1). Taking only the 2017 experiment into account, where C. rubiginosa and C. stigmatica could be compared directly because they were reared in identical conditions, there was a marginally significant difference in pupal developmental rates between the two species (GLS ANOVA, photoperiods  www.nature.com/scientificreports www.nature.com/scientificreports/ combined, effect of species: F 1,975 = 4.7, P = 0.03) but a highly significant temperature by species interaction (F 1,975 = 20.1, P < 0.00001). Short-day conditions slightly but significantly accelerated pupal development in both species ( Fig. 2c,g,h), although there did exist sporadic deviations from this general tendency in some experimental regimens (Tables 2 and 3). In addition, there was a significant temperature by experimental year interaction in the GLS model fit for C. stigmatica (Table 4), such that pupae in the 2018 experiment developed slower at low temperatures but faster at high temperatures than pupae in the previous year. In fact, lower temperature thresholds for pupal development differed more between experimental years in C. stigmatica than they did between photoperiods or even species (Tables 5 and 6).

Adult body mass.
Regardless of experimental conditions, females of C. rubiginosa and C. stigmatica were significantly heavier than males (Table 4, Fig. 3). The effect of developmental temperature on adult body mass was subtle and only marginally significant. In C. rubiginosa, body mass was significantly affected by photoperiod, which was manifested in a weak tendency for relatively heavier adults under long-day conditions ( Table 4, Fig. 3a). Across all temperature groups, C. rubiginosa males averaged at 15.8 mg under a short-day photoperiod and 16.4 mg under a long-day photoperiod; respective body mass values in females were 19.9 and 20.1 mg. In C. stigmatica, the effect of photoperiod on adult body mass was non-significant but there was a pronounced and significant difference between two experimental years (Table 4). Beetles reared on cut leaves in 2017 generally weighed more than those that emerged after developing on living plants in 2018 (Fig. 3b,c). On average across all experimental regimens in 2017, male C. stigmatica body mass was 11.0 mg and female body mass, 13.6 mg, whereas in 2018, mean male and female body mass only amounted to 10.1 and 12.1 mg, respectively. Even during the first experimental year, C. stigmatica was significantly smaller than C. rubiginosa (GLS ANOVA, effect of species: F 1,975 = 1710.0, P < 0.00001). The largest of C. stigmatica females weighed about 16 mg and were approximately the size of average C. rubiginosa males.

Discussion
Experimental rearing under different thermal and photoperiodic conditions reveals that immature survival, development and growth in tortoise beetles C. rubiginosa and C. stigmatica are sensitive to temperature and day length. However, the degree of this sensitivity varies considerably across species, developmental stage, and trait studied. Some of the responses were expected. For example, a sharp linear increase in developmental rate with rising temperature is a well-established phenomenon 16,46 . Acceleration of development by short-day conditions was also anticipated to occur as it is a widespread form of adaptive plasticity in response to seasonal time constraints. On the other hand, there are quite a few surprising findings which are more challenging to interpret. In particular, body mass in C. stigmatica and C. rubiginosa is largely independent of rearing temperature, contrary to a commonly observed negative size-temperature relationship. Small body size is often associated with rapid development but may alternatively be a consequence of disease, poor nutrition or stressful conditions, when development is prolonged. This is not the case here either, as C. stigmatica, which is significantly smaller than C. rubiginosa, develops significantly slower. Furthermore, C. stigmatica has even slower development and smaller body mass when reared under more favourable conditions. Temperature-dependent development. Egg development in C. rubiginosa and C. stigmatica proceeds at such a similar pace that it is not possible to separate these two species by egg development time at any of the temperatures tested. By contrast, pupal and especially larval development is faster in C. rubiginosa than C. stigmatica. The former species also has steeper thermal reaction norms in most cases (Fig. 2, Tables 5 and 6; Supplementary  Fig. S1), which implies accumulation of fewer degree-days. All this is intriguing because C. rubiginosa is larger than C. stigmatica during all developmental stages. Usually, it is bigger insects that develop more slowly [47][48][49][50][51] and accumulate a greater sum of degree-days 52 . Nevertheless, both life-history theory 49 and evolutionary experiments 53 show that selection can result in a faster body mass gain without increasing development time. Males of both species develop faster than females, albeit in C. stigmatica the difference is not statistically significant with our sample sizes. In any case, relatively faster male development is ubiquitous among the insects 54 .
In comparison with other leaf beetles (Chrysomelidae), whose thermal requirements for development are known, C. rubiginosa and C. stigmatica have slightly right-shifted lower developmental thresholds and smaller-than-average sums of degree days. In particular, an average studied leaf beetle requires 91.7 degree-days above 10   www.nature.com/scientificreports www.nature.com/scientificreports/ for pupal development 16 . More experiments with different tortoise beetle species are needed to find out whether relatively high threshold and slope values (the sum of degree-days equals 1/b) are typical of this whole group.

Temperature, sex, diet and body mass. Of all the factors studied, body mass in C. rubiginosa and C.
stigmatica is mostly influenced by species identity and sex (Fig. 3, Table 4). While the effect of temperature is marginally significant and it is possible to discern weak tendencies in Fig. 3, differences between thermal regimens are too small to discuss them meaningfully. This finding conflicts with the widespread but poorly understood "temperature-size rule", whereby most ectotherms attain larger size in cooler conditions 55 . Female beetles of both species are bigger than males, as is usual with insects 56 .
A smaller body mass in C. stigmatica is in line with hypotheses that animal species with narrower host plant ranges should comprise smaller individuals. However, opinions vary as to why this should be so and what comes first in such evolution: body size or diet breadth 47,57,58 . Also, while the latter two cited works do indeed show that larger moths have broader host plant repertoires, a study on butterflies 48 finds no such relationship. We are inclined to agree with Wasserman and Mitter 57 that a larger body size might enhance the generalists' ability to cope with environmental variation and physiological stress. Not only is C. rubiginosa larger and less host-specific than C. stigmatica, but it also has a wider distribution range and, as our experiments show, better survives at low temperatures (Fig. 1). However, as C. stigmatica feeds on members of the tribe Anthemideae that contain high concentrations of monoterpenes which are responsible for their strong aromatic odors and insecticidal properties 59 , its smaller body size may as well be due to a greater investment of energy into detoxification of host plant defensive chemicals. Interestingly, larval development on living host plants during the 2018 experiment resulted in even smaller adult body mass, especially in females (Fig. 3b,c), relative to the previous year's results when C. stigmatica was reared on cut leaves at a high relative humidity. The aim of the 2018 experiment was to improve rearing conditions and not to explicitly test for an inhibitory effect of host plant on herbivore development. It is not possible to quantify the confounding effects of genetic background and relative humidity which also differed between years. Nevertheless, it is worth noting that, while we did achieve higher survival rates, we obtained smaller adults with less pronounced sexual size dimorphism, which is actually indicative of poorer conditions 56 .
Photoperiodic plasticity of developmental rate and body mass. Short-day photoperiod accelerates larval and pupal development in C. rubiginosa and C. stigmatica, albeit not strongly and not at all temperatures (Fig. 2, Table 2-4). In nature, both species complete only one generation per year and hibernate in the adult stage 37,38,41 . Therefore, relative acceleration of development as the season is waning and day length is decreasing apparently ensures that the overwintering stage is reached before the onset of cold weather and deterioration of host plants. The effect is especially pronounced in C. stigmatica larvae, which have the longest development time and thus face a higher risk of maturation at a suboptimal time of the year. Besides, short-day larvae of C. stigmatica have significantly steeper thermal reaction norms (Fig. 2e,f), i.e., their developmental rate is more sensitive to temperature change, which may be important in taking advantage of spells of warm weather late in the season. In C. rubiginosa, development under short-day conditions is accomplished at a cost of increased larval mortality rates (Fig. 1b) and slightly but significantly reduced adult body mass (Fig. 3a). By contrast, C. stigmatica seems to be better adapted to late-season conditions, as its larval mortality is less dependent on day length, despite stronger developmental acceleration, and adults are not smaller under short-day conditions, perhaps owing to a more frugal metabolism. There is ample evidence that many temperate insects accelerate development and/or accumulate reserves for successful overwintering 22,25,60-62 . Repeatability of results. This is the first study to test the effects of temperature and photoperiod on the immature development in C. stigmatica, and we are not aware of any published material that could be compared with our findings. However, our experiments are carried out during two consecutive years and it is important to note that the main results are essentially replicated in the second year (Figs 2 and 3), in spite of different rearing conditions. Although C. rubiginosa was not previously studied in a similar experimental setting either, there exist estimates of its development time under laboratory and field conditions. www.nature.com/scientificreports www.nature.com/scientificreports/ Egg and pupal durations measured by us in C. rubiginosa are close to those reported by Ward and Pienkowski 11 , but larval development times and adult body masses differ dramatically. The population studied by these authors originated from Virginia (USA), and larvae were reared on potted thistles (in the present study, C. rubiginosa was reared on fresh cut leaves of burdock) under a 13-h and 17-h photophase. Despite higher survival rates, larval development in their experiments was slower (e.g., 15.2 days at 26.6 °C and 35 days at 17.8 °C -cf. Table 2) and adults were smaller (treatment group means ranged from 10.5 to 14.4 mg -cf. Fig. 3a). This discrepancy is intriguingly reminiscent of our findings with C. stigmatica reared on cut leaves vs. living plants, but it is premature to draw parallels because of many possible confounding factors involved. The same authors showed 11 that C. rubiginosa developed in the field significantly faster than could be expected from their experimental estimates.
Also in Virginia, Spring and Kok 8 found that the period from hatching to adult emergence under field conditions was about 22 days on thistles and 23.8 days on burdock. Mean temperature during that period was approximately 22 °C. Thus, development times recorded by Ward and Pienkowski 11 are anomalously long, whereas our laboratory estimates of postembryonic development time in a Russian population of C. rubiginosa fed with cut burdock leaves (Table 2) are in good agreement with the phenology of this species in Virginia. Body mass of freshly eclosed, unfed adults in our experiments (Fig. 3a) is also consistent with body mass of field-collected and overwintered adults from Virginia (17.6 and 24.4 mg for males and females, respectively 11 ). In addition, field development times and body masses suggest that defensive reactions from host plants, if any, do not significantly inhibit larval development and growth in C. rubiginosa. A possible alternative explanation for the discrepancy between the two rearing methods may be that potted and wild-growing plants differ in nutrient content. So far, the causes of prolonged development and small body size in both tortoise beetle species when reared on living plants in the laboratory remain unknown. Eggs were carefully excised with the underlying leaf fragment or, in the case of C. rubiginosa, which did not always oviposit on the leaves, detached from container walls using a sharp razorblade. Collected eggs were transferred with a moistened paintbrush to small plastic Petri dishes (40 mm in diameter), which in turn were placed into larger dishes (100 mm in diameter) on a layer of damp cotton wool to avoid desiccation of the eggs. Egg development was monitored daily, and, when leaf fragments deteriorated, fresh ones were added to prevent future hatchlings from starvation. On the day of hatching, each larva was carefully picked with a blunt preparation needle and transferred to a freshly cut leaf fragment that was placed into a 4 cm 3 plastic cup with ventilation holes in the plug and a moistened piece of paper towel on the bottom. Each plastic cup usually housed 3 or 4 larvae (seldom 2 or 5, which depended on the number of hatchlings available). Larvae and pupae were also checked daily. Food and paper towel were changed as needed. Pupae were detached using forceps and transferred to 250 ml plastic containers where they were laid on a moistened cotton wool layer.

Methods
Relative humidity in larval cups during the 2017 experiment was maintained close to 100%, which appeared to be suboptimal to C. stigmatica (see below). However, it was not possible to maintain a lower humidity because cut leaves quickly withered and dried out. Therefore, an attempt was made at rearing C. stigmatica larvae on living plants. In October of 2017, tansy seeds were collected in the natural habitat of this beetle in Bryansk. The seeds were dried at room temperature and stored at 4 °C. In March of 2018, the seeds were sown in commercially available potting soil in indoor boxes under Dulux L 55 W/830 fluorescent lamps (Osram GmbH, Germany). Early in May, seedlings were individually transplanted to 500 ml plastic glasses half-filled with potting soil and covered with a paper sheet that was fastened to the rim by means of a rubber band. Newly hatched larvae of C. stigmatica were transferred to these tansy seedlings, 6-10 larvae per plant, and kept there until pupation. In all other respects, the rearing procedure was the same as in 2017.
Experimental design. Eggs laid during the previous 24-h period were randomized among ten experimental regimens: five constant temperatures (16,19,22,25, and 28 °C) and two photoperiods (short-day 12 L:12D and long-day 18 L:6D). Throughout the entire immature development, all individuals remained in the regimens to which they had initially been assigned. Eggs laid by both species before the arrival to the laboratory in 2017 and all eggs of C. stigmatica in 2018 were incubated until hatching in the chamber where parental adults were kept (23-24 °C, 18 L:6D), and newly hatched larvae were allocated among the same ten experimental regimens. Eggs, larvae, and pupae were monitored daily, and the date and time of hatching, pupation and adult emergence were recorded. Adults were weighed on a Discovery DV215CD electronic balance with 0.01 mg precision (Ohaus Corporation, USA) on the day of eclosion. Sex was determined by dissection.
Temperature in the environmental chambers was maintained to ±0.1-0.5 °C via a software-controlled balance of heating and cooling (RLDataView 1.03; Research Laboratory of Design Automation, Taganrog, Russia) and automatically recorded every 10 s. Actual rearing temperatures slightly deviated from the set values and are given in Tables 2 and 3, but for convenience we refer to them as integers throughout the text. (2019) 9:10047 | https://doi.org/10.1038/s41598-019-46421-3 www.nature.com/scientificreports www.nature.com/scientificreports/ Statistical analyses. Statistical analyses were carried out in R version 3.5.1 with RStudio 63,64 . In all analyses, temperature was treated as a continuous independent variable and all other factors (photoperiod, sex and experimental year) were treated as categorical predictors. No random-effects structure could be identified because rearing groups consisted of randomly picked individuals. A preliminary inspection of data showed no relationship between rearing-group size and either development time or adult body mass, and so it was unlikely that group rearing or variation in survival rates could have introduced significant confounding to our results.
Survival of immature stages and adult sex ratio under experimental combinations of temperature and photoperiod were analyzed by fitting generalized linear models (GLMs) with a logit link and binomial error structure. In addition, for illustration purposes, survival rate in each regimen was expressed as a percentage of individuals successfully completing a given stage ± binomial s.e.
Egg, larval and pupal developmental rates were calculated for each individual as inverse durations of the corresponding stages (days −1 ). The effects of temperature, photoperiod, sex, experimental year and species identity on developmental rate and body mass were analyzed using the generalized least-squares (GLS) method under restricted maximum likelihood with different variances for each combination of factors 65 . Analyses were performed using the gls() function in the nlme package 66 . Significance of differences was determined with F-tests based on type I (sequential) sum of squares. Model assumptions of homoscedasticity, linearity, and normality of residuals were verified by inspection of raw and standardized residuals plots.
The responses of developmental rate (R) to temperature (T) were described in greater detail by means of linear regression equations of the form 46 R = a + bT. GLS models were re-run with temperature as the single explanatory variable for all subsets of data where the response significantly (p < 0.0001) differed between the levels of a categorical predictor. The intercept (a) and slope (b) were thus obtained separately for each combination of species, developmental stage, photoperiod and experimental year. These a and b values were then used to calculate two biologically meaningful parameters: the lower temperature threshold for development LTT = −a/b and the sum of degree-days SDD = 1/b.

Data Availability
The datasets generated during the current study are available as online Supplementary information.