CryoLetters 37 (3), 206-217 (2016) © CryoLetters, [email protected]

SUPERCOOLING CAPACITY AND CRYOPROTECTANTS OF OVERWINTERING LARVAE FROM DIFFERENT POPULATIONS OF HOLCOCERUS HIPPOPHAECOLUS

Bin Tian, Lili Xu, Miao Zhang, Yuqian Feng and Shixiang Zong*

Beijing Key Laboratory for Forest Pest Control, Beijing Forestry University, Beijing, 100083, P.R. China *Corresponding author e-mail: [email protected]

Abstract

BACKGROUND: Holcocerus hippophaecolus is the most serious pest occurred in seabuckthorn forest of three north areas. OBJECTIVE: The primary aims of the current study were to explore the physiological mechanisms and adaptability of H. hippophaecolus to low temperatures. MATERIALS AND METHODS: Assessing supercooling point, freezing point, and cryoprotectants of different larval instars from three different populations. RESULTS: Supercooling capacity of larvae from the 8–13 instar groups was relatively independent of temperature and other indicators such as latitude. Larvae from the 14–16 instar groups were sensitive to temperature and latitude, with generally lower limits and a wider range of SCPs than those of the other instar groups. CONCLUSION: For each population, the differences in the supercooling capacity of different instar stages for the identical period were not significant. The metabolism of fat and glycogen might not be the primary factors affecting the supercooling capacity. Keywords: Holcocerus hippophaecolus, overwintering larvae, different populations, different instars, supercooling point, cryoprotectant content

INTRODUCTION reported by Bachmetjew as discussed by Somme (4). The supercooling point (SCP) of an is are poikilothermic , and an important indicator of cold hardiness and a climate change is a primary factor that can strategy for overwintering and has been widely influence seasonal growth and cause declines in used to predict potential dispersal and populations. In both temperate and frigid geographic distributions of insect source regions, insects experience annual cold stress. populations. Therefore, cold hardiness is an important factor The supercooling capacity of insects is in the life history of insects in these areas, highly variable between both individuals and affecting reproduction, dispersal, distribution, groups, and the primary influences on and population dynamics in the following supercooling capacity are divided into external seasons (1) To adapt to low environmental and internal factors. The external factors temperatures, insects developed strategies to primarily include seasonal change, geographic safely overwinter in the long-term process of condition, and host-plant species, among others, evolution. Maintaining a supercooled state is a whereas the internal factors include the primary strategy for insects to overwinter in the biological state, developmental stage, and temperate and frigid regions of the Northern physiological and metabolic state of the body. Hemisphere (2). Additionally, the supercooling point in insects is Fahrenheit (1842) discovered supercooling affected by a change in the water ratio with the and found that water remains a liquid when the production and transformation of temperature falls below the freezing point (FP) cryoprotectants, particularly fats, low-molecular under certain conditions reported by Li (3). The weight substances, and polyhydric alcohols, phenomenon of supercooling in insects was first amino acids, and anti-freezing proteins (AFPs),

206 which are stimulated by changes in environment, will provide the theoretical basis to assess host, developmental stage, and biological state. overwintering strategies, bio-physiological The interaction of these external and internal regulation, and the potential for population factors regulates the supercooling capacity of establishment. insects (2). In the long life history of Holcocerus MATERIALS AND METHODS hippophaecolus (: ), 4 years are required to complete a generation, which Study sites and sample collection. commonly has 16 instars (5). The larva feeds on The specimens used in these experiments the trunk and roots of sea buckthorn. Newly were collected from the field in China during hatched larvae bore primarily into the phloem of mid-March 2013. The three different collection the trunk, which leads to desiccation of the bark. sites were Jianping in Liaoning Province (JP), A few larvae bore into the xylem and damage Zhungeerqin in Inner Mongolia (ZQ), and roots as they move underground to overwinter, Pengyang in Ningxia Hui Autonomous Region and, as the damage accumulates and most roots (PY; Table 1). All overwintering larvae were are hollowed out, the host eventually dies (6-10). transported in soil with roots of sea buckthorn to In recent years, most areas with sea buckthorn the laboratory at Beijing Forestry University experienced outbreaks of this species, including within 24 h without any treatment; the healthy the provinces of Shaanxi, Shanxi, Liaoning, and larvae were transferred to the laboratory for Hebei and Inner Mongolia and Ningxia Hui testing. The local temperature (°C) data were autonomous regions. The damaged area has obtained from the China Meteorological Data increased to 133,000 hm2, with over 67,000 hm2 Sharing Service System (http:// cdc.cma.gov.cn). in which the mortality of sea buckthorn is total. Table 1. Geographic location, altitude, and temperature conditions at the collection sites.

Collection Longitude Latitude Altitude Mean Jan. Mean Mar. Accumulated Low Site (°E) (°N) (m) Temp. (°C) Temp. (°C) Temp. (Days -°C) JP 119°54' 41°19' 441 -10.6 1.5 -952.3

ZQ 110°25' 39°52' 1169 -7.4 4.4 -798.0

PY 106°16' 35°50' 1788 -5.3 7.3 -455.2

The accumulated low temperature is the mean of the total subzero daily temperatures from November 2012 to March 2013. JP, JianPing in Liaoning Province; ZQ, Zhungeerqi in Inner Mongolia Autonomous Region; PY, Pengyang in Ningxia Hui Autonomous Region.

Moreover, damage caused by this species is Measurement of instars. increasing. The general research on H. All larvae were photographed under a hippophaecolus has concentrated primarily on microscope (LEICA EZ 4D). The head widths bio-ecological characteristics, population and body lengths of instars were measured, with dynamics, chemical prevention, sex the magnification noted(6). Based on the pheromones, mechanisms to explain outbreaks, distribution of measurements, three groups of and monitoring for early warning of outbreaks instars (8–10, 11–13, and 14–16) were tested. (11-18). Although the collective achievements are considerable, the cold hardiness of H. Measurement of SCP and FP. hippophaecolus from different populations has Ten or more larvae of each group of instars not been investigated. from each population were selected. Each larva The primary aims of the current study were was fixed to a thermocouple probe with to explore the physiological mechanisms and parafilm, and the thermocouple was connected adaptability of H. hippophaecolus to low to an automatic data recorder (an instrument to temperatures by assessing the supercooling measure the SCP). A larva was placed in a capability of different groups of larval instars cotton-lined container and transferred into a from three populations. The results of this study freezing chamber (high-low temperature test 207 chamber), and, at the cooling rate of 1°C/min, Measurement of glycogen content. the body temperature of the larva was recorded. Glycogen content was determined using the The temperature at which an exothermic phenol and concentrated sulfuric acid method response was first detected was the supercooling (20). Each larva was dried, homogenized in 10 point, and the FP was the point at which larval ml tubes with 70% ethanol, and then centrifuged temperature continued to decline following at 2,600 × g for 10 min three times before conclusion of the exothermic process. recovering the supernatant (21). The residue was mixed with 6 ml of 10% (vol:vol) trichloroacetic Measurement of water content. acid and heated at 70°C for 15 min in an To assess the water content, ten more larvae electrothermostatic water bath. The mixture were selected from each of the three groups of cooled and was then centrifuged for 15 min at instars from each of the three populations. The 3,000 × g. A UV spectrophotometer was used to fresh mass (FM) of an individual larva was determine the absorbance values of supernatant determined on an electronic balance (±0.001 g). extracts at 490 nm. Measurements were obtained The larva was then oven-dried at 60°C for 72 h using 5–10 larvae from each population of H. to determine dry mass (DM). The water content hippophaecolus. as a ratio was calculated using FW and DW in the following formula: (FM − DM)/FM × 100%. Statistical analyses. All statistical analyses were conducted Measurement of fat content. using the SPSS 19.0.0 statistical software The fat content was measured as described package. The mean SCPs and FPs and water, fat, by Liu et al. A dried larva was homogenized, and glycogen contents were analyzed and and the fat content was extracted using a mixture compared with one-way ANOVA (α = 0.05) and of chloroform and methanol (19) in the least significant difference tests (LSD, α = 0.05). following steps. A dried larva was ground to Pearson’s correlations were used to assess the powder in a mortar. The powdered, dried larva relationships between the SCPs and FPs and the was weighed (DW), placed in a 10 ml tube, and geographical, climatic, and physical data. homogenized with 4 ml of a methanol:chloroform mixture (methanol:chloroform = 1:2). The supernatant RESULTS was filtered following centrifugation at 2,600 × SCP and FP analyses. g for 10 min, and then 4 ml of The SCP and FP both differed significantly methanol:chloroform was added to the residue. among the three populations of H. This process was repeated twice. For the hippophaecolus (Fig. 1). The mean SCPs of 8– remaining residue, the lean dry weight (LDW) 10 and 11–13 instar groups in the ZQ population was determined after baking at 60°C for 72 h in were significantly lower than those in the PY (P an oven. The fat content was calculated with the 1 < 0.010, P = 0.037) and JP (P < 0.010, P = following formula: fat content = [(DW − 2 1 2 0.018) populations. In 14–16 instar groups, the LDW)/FW] × 100. Measurements were obtained mean SCP of the JP population was the lowest for 6–18 larvae from each of the populations of and was significantly lower than that of the PY H. hippophaecolus. (P = 0.04) population. In all groups of instars, the mean FP of the PY population was higher than that of the ZQ (P < 0.05) and JP (P < 0.05)

populations.

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The distributions of SCPs were different for and the narrowest and smallest were in the 8–10 H. hippophaecolus larvae from the three instar group of the JP population. The highest populations (Fig. 2). In the 8–10 instar group, SCP value was in the 14–16 instar group of the the range and coefficient of variation for the ZQ population, and the lowest value was in the SCPs of JP larvae (CV = 14.82, range = 1.50) 14–16 instar group of the PY population. In were lower than those of the PY (CV = 28.36, analyzing the distributions of 8–10 and 14–16 range = 2.50) and ZQ (CV = 21.53, range = instar groups, for both groups, the ZQ 4.20) larvae. The highest SCP value appeared in population had the widest range and the most PY larvae (−2.10°C), and the lowest value was extreme values and the JP population had the found in ZQ larvae (−6.40°C). In the 11–13 narrowest range with the least extreme values. instar group, the range and coefficient of For 11–13 instar groups, the differences in variation for the SCPs of PY larvae (CV = 29.84, ranges were consistent with variations in range = 2.70) were lower than those of the ZQ temperature and longitude; JP had the widest (CV = 21.49, range = 3.10) and JP (CV = 28.51, and PY had the narrowest range. range = 4.20) larvae. The highest and lowest The distributions of FPs were different for values were both found in JP larvae (−1.90°C H. hippophaecolus larvae from the three and −6.10°C, respectively). In the 14–16 instar populations (Fig. 2). In the 8–10 instar group, group, the range and coefficient of variation for the range and coefficient of variation of the FPs the SCPs of JP larvae (CV = 26.44, range = in the ZQ larvae (CV = 24.89, range = 2.10)

Figure. 1 SCPs and FPs of three instar groups of H. hippophaecolus larvae in three populations. Note: Different capital and lowercase letters below the bars indicate significant differences among SCPs and FPs, respectively (P < 0.05). 3.30) were lower than those of the PY (CV = were larger than those of the PY (CV = 21.79, 37.07, range = 4.30) and ZQ (CV = 38.98, range range = 0.90) and JP (CV = 18.61, range = 1.50) = 5.50) larvae. The highest SCP value was found larvae. The highest FP value was found in ZQ in PY larvae (−1.50°C), with the lowest value in larvae (−0.80°C), and the lowest value was ZQ larvae (−7.90°C). For all instar groups, the found in both ZQ and JP larvae (−2.90°C). In widest range and largest CV for SCPs were in the 11–13 instar group, the range and coefficient the 14–16 instar group of the ZQ population, of variation of the FPs in the JP larvae (CV =

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25.56, range = 2.30) were larger than those of and 14–16 instar groups of the PY population the PY (CV = 29.20, range = 1.30) and ZQ (CV and in the 8–10 instar group of the ZQ = 28.51, range = 4.20) larvae. The highest FP population; the lowest FP value was found in the value was found in PY larvae (−0.80°C), and the 14–16 instar group of the PY population. In lowest value was found in JP larvae (−3.60°C). analyzing the distributions of 8–10 and 14–16 In the 14–16 instar group, the range and instar groups, for both groups, the ZQ coefficient of variation of the FPs in the JP population had the widest range and the most larvae (CV = 22.50, range = 1.60) were lower extreme values and the JP population had the than those of the PY (CV = 43.70, range = 2.60) narrowest range with the least extreme values. and ZQ (CV = 35.80, range = 3.40) larvae. The For 11–13 instar groups, the differences in highest FP value was found in PY larvae ranges were consistent with variations in (−0.80°C), and the lowest value was found in temperature and longitude; JP had the widest ZQ larvae (−4.60°C). For all instar groups, the and PY had the narrowest range. When the widest range was in the 14–16 instar group of distributions of SCPs and FPs of the instar the ZQ population, which also contained the groups were compared, the ranges of FPs of the lowest FPs, and the narrowest range was for the 11–13 instar groups were similar to those for the 8–10 instar group in the PY population. The 14– SCPs of the identical instar groups. 16 instar group of the PY population was the The SCPs and FPs of the different instar most variable, and the 8–10 instar group of the groups are shown in Fig. 4. For the SCPs, we JP population had the least variation. The found differences among the instars of the JP highest FP value was found in both the 11–13 population; the SCP of the 14–16 instar group

Figure 2. Distribution of SCPs of the different instar groups of H. hippophaecolus larvae from three populations. 210

was significantly lower than that of the 11–13 instar group, which included the lowest value instar group (P = 0.020), but the SCPs of 8–10 (−7.90°C), and the narrowest range was in the and 11–13 instar groups were not significantly 11–13 instar group. The highest value was different. The differences in SCPs among the observed in the 8–10 instar group (−2.20°C). In different instar groups of the other two the PY population, the widest range was in the populations were not significant. The FPs of the 14–16 instar group, which included both the 14–16 instar group in both the JP (P = 0.020) highest (−1.50°C) and lowest (−5.80°C) values, and the ZQ (P = 0.018) populations were and the narrowest range was in the 8–10 instar significantly lower than those of the other two group. For all instar groups, the range of SCP instar groups. In the PY population, the FPs values for older overwintering larvae was wider were not significantly different among the instar than that for younger ones; in the PY population, groups. the range of SCP values was significantly wider The distributions of SCP values for the in the later instars. Although the highest SCP three instar groups were different (Fig. 2). In the values were found in different instar groups in JP population, the widest range was found for the three populations, the lowest values were all the 11–13 instar group, which included the observed in the 14–16 instar group of the three highest (−1.90°C) and lowest (−6.10°C) values; populations. the lowest value was also found in the 14–16 The distributions of FP values for the three instar group. The narrowest range of SCP values instar groups were different (Fig. 3). In the JP was in the 8–10 instar group. In the ZQ population, the widest range of FP values was in population, the widest range was in the 14–16 the 11–13 instar group, which included the

Figure 3. Distribution of FPs of the different instars of H. hippophaecolus larvae from three populations.

211 highest value (−1.30°C); the lowest value was Water content. observed in the 14–16 instar group (−1.80°C). The water contents of larvae from the three The narrowest range was in the 8–10 instar populations were significantly different (Fig. 5). group. In the ZQ population, the widest range of For instar groups combined, the water content FP values was in the 14–16 instar group, which was lowest in the ZQ population and highest in included the lowest value (−4.60°C); the highest the PY population. In 8–10 instar groups, the value was observed in the 8–10 instar group water contents of larvae from the three (−0.80°C). The narrowest range was in the 11– populations were not significantly different (JP, 13 instar group. In the PY population, the widest 56.82% ± 1.28%; ZQ, 54.88% ± 1.13%; and PY, range of FP values was in the 14–16 instar 57.96% ± 1.27%). In 11–13 instar groups, the group, which included both the highest water contents of the PY larvae (60.51% ± (−0.80°C) and the lowest (−3.40°C) values; the 1.27%) were significantly higher than those of highest FP value was also observed in the 11–13 the JP (54.96% ± 1.10%, P = 0.040) and ZQ instar group. The narrowest range was in the 8– (53.13% ± 1.29%, P = 0.180) larvae. In 14–16 10 instar group. The differences in the FPs were instar groups, the water contents of the PY identical to those in the SCPs. larvae (54.74% ± 1.79%) were higher than those Based on Pearson’s correlations, the mean of the ZQ larvae (50.12% ± 0.64%, P = 0.030), SCPs and FPs of the 8–10 and 11–13 instar and no difference was observed in water groups were not correlated with longitude, contents between JP larvae (54.15% ± 1.38%) latitude, altitude, accumulated low temperature, and those of the other two populations. or the mean temperatures of January and March. For 14–16 instar groups, the mean SCP was significantly correlated with latitude (r = −1.000, P = 0.009) and accumulated low temperature (r = −1.000, P = 0.009); the mean FP was not correlated with any index.

Figure. 5 Water ratio of different instars of H. hippophaecolus larvae in three populations. Note: Different lowercase letters above the bars indicate significant differences (P < 0.05).

The water contents of larvae from the different instar groups of each population are shown in Fig.6. In both the JP and ZQ populations, the water content declined with advancing instars. Although the decline in water content was not significantly different among the three instar groups in the JP population, the water content of larvae in the 8–10 instar group

Figure 4. SCPs and FPs of the different instars of H. hippophaecolus larvae in three populations. Note: Different capital and lowercase letters below the bars indicate significant differences 212 Figure 6. Water ratios of three populations of among SCPs and FPs, respectively (P < 0.05). H. hippophaecolus larvae in different instars. Note: Different lowercase letters above the bars indicate significant differences (P < 0.05). was significantly higher than that in the 14–16 0.001). In 14–16 instar groups, the fat contents (P = 0.013) instar group in the ZQ population. In of the ZQ larvae (0.54 ± 0.07 mg) were the PY population, the water content of larvae in significantly higher than those of the PY (0.31 ± the 11–13 instar group was significantly higher 0.03 mg, P = 0.001) and JP (0.02 ± 0.00 mg, P < than that in the 14–16 (P = 0.041) instar group. 0.001) larvae, and the fat contents of the PY The SCPs were not correlated with the larvae were significantly higher than those of the mean water contents of the larvae in the three JP population (P < 0.001). populations, three instar groups, or instar groups The fat contents of larvae in different instar within the different populations. groups of each population are shown in Fig. 8. Within each of the three populations, the fat Fat content. contents of larvae were not significantly The fat contents of larvae of the three different between the 11–13 and 14–16 instar populations were significantly different (Fig. 7). groups. However, the fat contents of larvae For all instar groups, the larvae in the ZQ increased significantly between the 8–10 and population had the highest fat content, and the 11–13 instar groups in both the ZQ (P < 0.001) larvae in the JP population had the lowest. 1 and the PY (P = 0.022) populations. In the JP Specifically, in 8–10 instar groups, the fat 2 population, the fat content declined significantly from the 8–10 to the 11–13 instar group (P = 0.001). The mean SCPs were not correlated with the mean fat contents of larvae in the three populations, three instar groups, or instar groups within the different populations. Glycogen content. The glycogen contents of larvae from the three populations differed significantly (Fig. 9).

Fig. 7 Lipid content of different instars of H. In 8–10 instar groups, the glycogen contents of hippophaecolus larvae in three populations. larvae from the JP (12.32 ± 2.77 mg), ZQ (11.43 Note: Different lowercase letters above the ± 2.61 mg) and PY (6.57 ± 0.99 mg) populations bars indicate significant differences were not different. In 11–13 and 14–16 instar (P < 0.05). groups, the glycogen contents of larvae from the contents of the ZQ larvae (0.27 ± 0.02 mg) were JP (4.31 ± 0.51 mg, P = 0.03; 2.68 ± 0.33 mg, P significantly higher than those of the PY (0.21 ± = 0.025, respectively) and PY (2.72 ± 0.28 mg, 0.02 mg, P = 0.001) and JP (0.07 ± 0.01 mg, P < P < 0.001; 2.64 ± 0.57 mg, P = 0.027, 0.001) larvae, and the fat contents of the PY respectively) populations were significantly larvae were significantly higher than those of the lower than those from the ZQ population (7.00 ± JP population (P < 0.001). In 11–13 instar 0.71 mg; 5.43 ± 1.53 mg, respectively). groups, the fat contents in the ZQ larvae (0.49 ± The glycogen contents of larvae in different 0.06 mg) were significantly higher than those in instar groups within each population are shown the PY (0.36 ± 0.06 mg, P = 0.049) and JP (0.03 in Fig. 10. For each population, the glycogen ± 0.00 mg, P < 0.001) larvae, and the fat content declined with the stages of instar. The contents of the PY larvae were significantly higher than those of the JP population (P <

Figure 9. Glycogen content of different instars of H. hippophaecolus larvae in threepopulations. Figure 8. Lipid content of three populations of 213 Note: Different lowercase letters above the bars H. hippophaecolus larvae in different instars. indicate significant differences (P < 0.05). Note: Different lowercase letters above the bars indicate significant differences (P < 0.05). glycogen contents of larvae in the 11–13 instar SCPs of H. hippophaecolus overwintering larvae groups were significantly lower than those in the in 14–16 instar groups declined with latitude (r 8–10 instar groups in both the JP (P = 0.006) = −1.000, P = 0.009), but a similar decline was and the PY (P = 0.001) populations. In the ZQ not apparent for the other two instar groups. In population, the fat content of different larval the JP population, although located with the instar groups was not significantly different. lowest mean monthly temperature, the The mean SCPs were not correlated with supercooling capacity was not stronger than that the mean glycogen contents of larvae in the in the other two populations. Therefore, latitude three populations, three instar groups, or instar and temperature might not be the primary factors groups within the different populations. affecting the supercooling capacity of the three populations. The cold hardiness of insects is affected by temperature either through long- or short-term acclimatization. Insect resistance to low temperature stress may be the result of a long- term process that developed primarily to prevent indirect injury from cold and freeze damage, whereas insects can withstand direct injury and shock from the cold after acclimation for a couple of hours or even tens of minutes (31). The difference in cold hardiness of insects from different populations may be affected by long- Figure 10. Glycogen content of three term climatic change and acclimation to the cold populations of H. hippophaecolus larvae in within a habitat. In this study, the mean SCPs different instars. were significantly correlated with latitude and Note: Different lowercase letters above the bars indicate significant differences (P < 0.05). accumulated subzero temperature from November 2012 to March 2013; therefore, the supercooling capacity of overwintering mature DISCUSSION larvae from the three populations was sensitive to changes of latitude and temperature. Population differences of the SCPs and FPs of However, for most instars (8–13) of H. H. hippophaecolus larvae. hippophaecolus overwintering larvae, the The supercooling capacity of insects is supercooling capacity was not associated with affected by many factors, including the climatic change. These results also environmental change and geographic demonstrated that for H. hippophaecolus distribution(22); whereas, the cold hardiness of overwintering larvae a decrease in the SCP insects is related to variation in the geographic might not be the most important strategy as a environment. Entomologists and ecologists response to cold stress. explore the role of supercooling capacity in the dispersal of populations with analyses of the Instar differences in the SCPs and FPs of H. geographic distributions of SCPs (23-26). In hippophaecolus. general, the supercooling capacity is stronger in The supercooling capacity is different for insects from high latitudes (altitudes) (27). For different developmental stages of insects. For example, the cold hardiness of Hemiberlesia example, Renlu (32) found that the SCPs of pitysophila Takagi in southern China increases pupae of the oriental fruit fly (Bactrocera significantly with an increase in latitude (28), dorsalis) are significantly lower than those of and the supercooling capacity of a wheat midge other developmental stages, and the SCPs of also tends to increase with latitude (29). Chinese citrus fly (Bactrocera minax) pupae are Additionally, the SCPs and FPs of diapause different from those of mature, diapause, and larvae of the Asian corn borer decline with an nondiapause larvae (33). The cold hardiness of increase in latitude (30). In this study, the three Artemia is also different among developmental populations were all from northern China, and, stages; the nauplius larval stage has the strongest although the altitude increased with a decrease cold hardiness, which is followed by adults. The in longitude, the difference in latitude among the other larval stage has the weakest cold hardiness three sites was not significant. In this study, the

214 and was sensitive to all experimental conditions to explore the lower limit of the SCPs. (34). Therefore, from a cold hardiness strategy, the On one hand, in comparing the FPs of overwintering larvae of H. hippophaecolus larvae from the three populations, a significant decline FP rather than SCP gradually to adapte decline was observed in later instars in our to cold stress during development. By inference, research. The FPs of mature larvae in the JP and the SCP of H. hippophaecolus larvae did not ZQ populations were significantly lower than decrease to increase cold hardiness, which is those of the other two instar groups. Thus, it consistent with the conclusion above. In further may be that the upper limit of freezing inside the investigations, the focus must be on the survival body declines for older instars of H. of H. hippophaecolus overwintering larvae in a hippophaecolus overwintering larvae from the frozen state. three populations. Role of cryoprotectants in the supercooling On the other hand, unlike the declining capacity of H. hippophaecolus larvae. trend with instars in the JP population, the SCPs The supercooling capacity of insects is a of the 11–13 instar groups from the ZQ and PY consequence of physiological and biochemical populations were lower than those of the other controls inside the body during adaptation to the two instar groups, and the differences among the environment. Based on previous research, water three instar groups were not significant. The and fat contents regulate changes in the SCP for reasons for this phenomenon may be associated most overwintering insects (36). An increase in with the stability of mid-instar larvae. In the cold hardiness following a decline in body water studies of Guo, the SCPs of Fopius arisanus content has been widely demonstrated for pupae at a mid-developmental stage are overwintering insects (37-39). In our research, significantly lower than those of other pupae and no relationship was found between the mature larvae; the mature larvae are ready for supercooling capacity of H. hippophaecolus transformation and therefore are sensitive to low overwintering larvae and water content. temperature, whereas 5-d-old pupae, a mid- However, changes in water contents of larvae developmental stage with organ differentiation are associated with the growth condition of the completed, are resistant to cold stress (35). In host and the amount of precipitation in the this study, the differences were not significant habitat (40, 41). among the three instar groups in the ZQ and PY Metabolites of fat are directly implicated in populations. A possible explanation could be the mediation of cold hardiness. For example, that different instars of overwintering larvae of cold hardiness of overwintering larvae of the H. hippophaecolus, with one generation in 4 Italian bee is closely correlated with the water, years, suffered from identical cold stress for one protein, and free-fat contents. In a study by period, which would result in identical cold Wang, the consumed protein of Trichogramma hardiness for overwintering. For a single dendrolimi is transformed to glycerol to increase population, the differences in the supercooling cold hardiness during diapause (42). In this capacity of different instar stages for the study, the fat contents of larvae from the three identical period were not significant. With a populations were different. Moreover, within vertical comparison of the range of variation and each of the populations, regular differences were the temperatures of the SCPs and FPs from the observed, which might be associated with the three instar groups within each population, we nutritional status of the host in different periods. found that the range was wider for later instar The changes in the fat contents of instar groups groups than for the younger group; however, the of the three populations were consistent. lower limit of SCPs all occurred in 14–16 instar Therefore, overall, the fat contents of larvae groups, which demonstrated that older larvae from the ZQ population were higher than those had a wider range than that of younger larvae to from the PY population, and the lowest fat adjust to climate change. content for each instar group was in the JP The trend in the variation of FPs in the population. However, the fat contents were three instar stages was similar to that of the significantly correlated with the SCPs (r = SCPs. However, the differences among the SCPs 0.742, P = 0.011) in the JP population, which were not greater than those of the FPs; thus, the suggested an important role for fat in controlling overwintering larvae of H. hippophaecolus the supercooling capacity of these larvae. broadened the range of the SCP as soon as the FP was extended. However, it was not obvious

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Based on many literature reports, the hippophaecolus larvae, which may facilitate factors that mediate cold hardiness in insects are studies with practical goals, such as most likely low-molecular weight sugar investigating overwinter survival, predicting alcohols, polyols, and AFPs. Low-molecular population dynamics of H. hippophaecolus weight sugar alcohols can enhance the cold larvae, and controlling population. Further hardiness of insects directly by increasing the investigations are required to explore the hemolymph concentration and stabilizing overwinter strategy and the relationship between membranes and proteins (43,44). In this study, the SCP of other developmental states and cold the glycogen contents and the mean SCPs of hardiness for insight into the adaptive larvae were not correlated, which indicated that mechanisms of H. hippophaecolus based on glycogen, as an energy storage substance, had no geographic differences effect on mediating the supercooling capacity of H. hippophaecolus overwintering larvae. The Acknowledgements: The National Natural differences in patterns of glycogen contents in Science Foundation of China (Grant No. the different populations and instar stages were 31470651) supported this study. likely explained in part by the growth condition and nutritional status of hosts of different age REFERENCES and from different habitats (45). Additionally, the larvae were sampled in March, which is a 1. McDonald JR, Head J, Bale JS & Walters K transitional period between seasons when FA (2000) Physiological Entomology. 25(2): temperatures increase rapidly. However, the p. 159-166. activity of H. hippophaecolus larvae would 2. Zhang R & Ma J (2013). Tianjin recover gradually, and, with this gradual Agricultural Sciences. 19(11): p. 76-84. recovery, the metabolism of fat and glycogen 3. Li BX, Chen Y L, & Cai HL (1998) would be activated. Thus, when the larvae were Entomological Knowledge. 35(6): p. 361- sampled, the fat and glycogen were not used to 364. resist cold stress. Similarly, the water content 4. Sømme L (1999) European Journal of did not reliably explain the differences in Entomology. 96(1): p. 1-10. supercooling capacity, because water directly 5. Hua BZ, Zhou Y, Fang DQ & Chen SL participates in various metabolic activities in this (1990) Entomography of China Carpenter period of transition. To explore the roles of the . Beijing: TianZe Press. three cryoprotectants in mediation of the 6. Zong SX, Luo YQ, Xu ZC & Wang T supercooling capacity and cold hardiness of H. (2006) Chinese Bulletin of Entomology. hippophaecolus overwintering larvae, the 43(5): p. 626-631. normal changes in physiological indexes in 7. Lu CK, Zong SX, Luo YQ, Xu ZC, Ma CD larval bodies for different overwintering periods & Zhao HY (2004) Journal of Beijing must be investigated further. Forestry University. 26(2): p. 79-83. In conclusion, the supercooling capacity of 8. Luo YQ, Lu CK & Xu ZC (2003) Forest H. hippophaecolus overwintering larvae from Pest and Disease. 22(5): p. 25-28. the three populations was different, although 9. Luo YQ, Lu CK & Xu ZC (2003) The among the three instar groups from each Global Seabuckthorn Research and population the supercooling capacity was Development.(1): p. 31-33. similar. For the larvae in 8–13 instar groups, the 10. Tian RM & Tang MC (1997). Inner supercooling capacity was relatively Mongolia Forestry Science & Technology. independent of temperature and other factors of (1): p. 36-38. the geographic environment, such as latitude. 11. Li J, Zhou J, Sun RB, Zhang HL, Zong SX, However, the larvae in 14–16 instar groups were Luo YQ, Sheng X & Weng Q (2013).. sensitive to temperature and latitude, with a Archives of Insect Biochemistry and tendency to a lower limit and a wider range of Physiology. 82(4): p. 183-195. SCPs than other instar groups. The effects of 12. Tao J, Chen M, Zong SX, & Luo YQ (2012) water, fat, and glycogen on the regulation of Plos One, 7(1). supercooling capacity of H. hippophaecolus 13. Wang R, Zhang L, Xu LL, Zong SX & Luo overwintering larvae were not obvious in this YQ (2015). Neotropical Entomology, 44(1): study. This study provides baseline information p. 68-76. on the overwintering strategy of H.

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