作者：刘培党，林兴凤，王大勇
【摘要】 目的:探讨热激胁迫对于涡虫发育以及脂肪和糖原积累的影响。方法:涡虫的发育通过体长与体质量来评价。分析35 ℃热激胁迫处理涡虫的体长与体质量变化以及实质细胞中脂肪与糖原积累情况，并评价热激胁迫处理涡虫中发育与脂肪或糖原积累之间可能的相关性。结果:与未经历热激处理的对照相比，热激胁迫处理不同长度时间(16、20、24、28与32 h)后涡虫体长与体质量显著减少或降低。与对照相比，热激胁迫处理导致涡虫实质细胞中的脂肪颗粒数量明显降低、尺寸显著变小。类似地，热激胁迫处理还导致涡虫实质细胞中的糖原积累的显著降低。进而，线性回归分析表明热激胁迫处理涡虫的体长与体质量均显著相关于实质细胞中脂肪颗粒的尺寸或糖原的含量。结论:热激胁迫处理可以明显抑制涡虫的发育，且热激胁迫处理涡虫中存在发育与脂肪或糖原积累之间的显著关联。
【关键词】 热激胁迫; 脂肪; 糖原; 发育; 涡虫
Freshwater freeliving planarians， an important component of the aquatic ecosystem， are distributed worldwide in unpolluted streams. Planarians have a high regenerative capacity， and every fragment is capable of growing into a complete inpidual if an inpidual planarian is cut into small fragments. Traditionally， planarians have been a favored model animal in developmental biology. They can be easily collected in large numbers， require only low culture and test medium volumes， and can be kept inexpensively in laboratory for the study of stem cell biology[1]， neuroregeneration[2]， and toxicology[3].
So far， the characteristics of planarian make it a suitable organism for studying the effects of environmental stresses. The acute toxicities of eight widely used surfactants on oxidative stress and cholinesterase(ChE) activities were examined in planarian Dugesia japonica， and the differences in acute toxicity among these surfactants were at least in the range of three orders of magnitudes[3]. The catalase activity was significantly induced by copper exposure at concentrations of 40， 80， and 160 μg·L1， suggesting that the catalase levels in planarians could represent a biomarker for estimation of copper effects in freshwater ecosystem[4]. The effects of UVirradiation to two planarian species were also determined， and UVrays caused obvious alterations of mortality， behavior， and morphology in each species[5]. In addition， the number of neoblasts extremely increased in the area of damaged tissue， immediately after UV irradiation[5]. Hypertonic osmotic shock treatment could induce the temporarily increase of putrescine and spermidine levels[6]. Moreover， the genes required for autophagy can function at the interface between survival and cell death during stressinducing processes like regeneration and starvation in sexual and asexual races of planarians[7].
The effects of low temperature on reproduction and regeneration have been investigated in planarians. The regeneration of planarians was significantly retarded for all levels by lowering the temperature[8]. Vowinckel(1970) showed that exposure to low water temperatures stimulated the increase in germ cells associated in testicular primordial and that this activity could be transmittable via homogenates[9]. In addition， the putrescine and spermidine levels could be temporarily increased after coldshock treatment in planarians[6]. Nevertheless， the possible effects of heatshock on the planarian development are still largely unclear.
Considering the fact that complex carbohydrates， such as lipid and glycogen， are involved in multiple biological processes in organisms， the first objective of this study was to investigate the effects of heatshock on development of planarians， and the second objective of this study was investigate the alterations of lipid and glycogen in heatshock treated planarians. Moreover， the possible associations of development with lipid or glycogen storage were examined in heatshock treated planarians.
1 Materials and methods
1.1 Animals
The planarians(Dugesia japonica) were collected from the stream in the Zijin Mountain， Nanjing， in 2009. The planarians were asexually maintained in autoclaved water at 22 ℃， and fed with chicken liver twice a week. Planarians were starved for more than 1 week prior to the experiments. The planarians were treated with heatshock at 35 ℃ for 16， 20， 24， 28， and 32 h. The planarians with the approximately same size(5 mm length) were used for assay of development and accumulation of lipid and glycogen.
1.2 Body weight
To calculate the average body weight of planarians， 10 animals were weighed after the water on the surface of animals was cleaned with filter paper. The experiments were repeated six times.
1.3 Body length
The midline length of planarians was examined to represent the body length. The experiments were repeated six times.
1.4 Lipid staining
A modified sudan black B staining method was used to evaluate the lipid accumulation in parenchymal cells as previously described[10]. Planarians were fixed in Bouins solution for 8 h， and the frozen sections were cut at 8～12 μm using freezing microtome(Leica CM1900， Germany). The sections were stained with 10 mg·ml-1 sudan black B solution， and the stained lipid droplets were dark blue. The diameters of stained lipid droplets were calculated to reflect the size of lipid droplet， and the experiments were repeated at least three times.
1.5 Glycogen staining
The periodic acidschiff(PAS) method for glycogen staining in parenchymal cells was performed as previously described[11]. Planarians were fixed in Bouins solution for 8 h， and the frozen sections were cut at 8～12 μm using freezing microtome(Leica CM1900， Germany). Sections were treated with 0.5% periodic acid for 10 min， rinsed in water for 10 min， and stained with schiff reagent for 10 min. Moreover， the sections were rinsed with sodium pyrosulfite for 2 min， washed in water for 5 min， and dehydrated and mounted in neutral balsam. Control sections were pretreated with aamylase. The stained glycogen was red color in the cytosol. The intensities of stained glycogen were examined， and the experiments were repeated at least three times.
1.6 Statistical analysis
All data in this article were expressed as means ± S.D. Graphs were generated using Microsoft Excel(Microsoft Corp.， Redmond， WA). Oneway analysis of variance(ANOVA) followed by a Dunnetts ttest was used to determine the significance of the differences between the groups. The probability levels of 0.05 and 0.01 were considered statistically significant. The associations of body weight with accumulation of lipid or glycogen， as well as the associations of body length with accumulation of lipid or glycogen， were examined with the method of linear regression in heatshock treated planarians.
2 Results
2.1 Effects of heatshock on body weight of planarians In the present study， the development of planarians was evaluated by the endpoints of body weight and body length. We first investigated the effects of heatshock on body weight. As shown in Fig 1， after heatshock(35 ℃) treatment for different time intervals(16， 20， 24， 28， and 32 h)， the body weights of heatshock treated planarians were all significantly(P&<0.01) decreased compared with those in control planarians without heatshock treatment.
Bars represent means ± S.D. Control， without heatshock treatment. a. P&<0.01 vs control
Fig 1 Effects of heatshock on body weight of planarians2.2 Effects of heatshock on body length of planarians We next examined the effects of heatshock on body length. As shown in Fig 2， similarly， after heatshock treatment for different time intervals， the body lengths of heatshock treated planarians were also all significantly(16 h， P&<0.05; 20， 24， 28， and 32 h， P&<0.01) decreased compared with those in control planarians without heatshock treatment. Therefore， treatment with the stress of heatshock can obviously suppress the development of planarians.
Bars represent means ± S.D. Control， without heatshock treatment. a. P&<0.05 vs control， b. P&<0.01 vs control
Fig 2 Effects of heatshock on body length of planarians
2.3 Effects of heatshock on lipid accumulation in planarians Again， we examined the effects of heatshock treatment on lipid accumulation in planarians. As shown in Fig 3A， compared with the lipid accumulation in control planarians without heatshock， heatshock treatment resulted in the sharp decrease of number of lipid droplets in parenchymal cells. Moreover， after heatshock treatment for different time intervals， the relative size of lipid droplets in parenchymal cells of heatshock treated planarians were significantly(P&<0.01) reduced compared with those in control planarians without heatshock treatment(Fig 3B). Therefore， treatment with the stress of heatshock will inhibit the lipid accumulation in parenchymal cells of planarians.
2.4 Effects heatshock on glycogen accumulation in planarians Considering the fact that glycogen is another important carbohydrate in living organisms， we further investigated the effects of heatshock treatment on glycogen accumulation in planarians. As shown in Fig 4， after heatshock treatment for different time intervals， the relative intensities of labeled signals for glycogen in parenchymal cells were significantly(P&<0.01) decreased compared with those in control planarians without heatshock treatment. Therefore， treatment with the stress of heatshock will also suppress the glycogen accumulation in parenchymal cells of planarians.
A. The pictures of lipid accumulation in parenchymal cells of control and 24 h heatshock treated planarians. B. Comparison of the relative sizes of lipid droplets in parenchymal cells between control and heatshock treated planarians. Control， without heatshock treatment. a. P&<0.01 vs control
Fig 3 Effects of heatshock on lipid accumulation in planarians 2.5 Associations of development with the accumulation of lipid or glycogen in heatshock treated planarians Linear regression analysis further suggests that the body weight was significantly(P&<0.01) correlated with the relative size of lipid droplet(R2=0.787)， and the relative intensity of glycogen signal(R2=0.814) in heatshock treated planarians(Tab 1). Similarly， the body length was significantly(P&<0.01) correlated with the relative size of lipid droplet(R2=0.801)， and the relative intensity of glycogen signal(R2=0.884) in heatshock treated planarians(Tab 2). Therefore， the close association between the development and the accumulation of lipid or glycogen exists in heatshock treated planarians.
A.The pictures of glycogen accumulation in parenchymal cells of control and 24 h heatshock treated planarians. B. Comparison of the relative intensities of labeled signals for glycogen in parenchymal cells between control and heatshock treated planarians. Control， without heatshock treatment. a. P&<0.01 vs control
Fig 4 Effects heatshock on glycogen accumulation in planarians Tab 1 Associations of body weight with the accumulation of lipid or glycogen in heatshock treated planarians as assayed by linear regression analysis
DependentIndependentPartial R2Pvariablevariablebody weightRelative size of lipid droplet0.787&<0.01Relative intensity of glycogen signal0.814&<0.01
Tab 2 Associations of body length with the accumulation of lipid or glycogen in heatshock treated planarians as assayed by linear regression analysis
DependentIndependentPartial R2Pvariablevariablebody weightRelative size of lipid droplet0.801&<0.01Relative intensity of glycogen signal0.884&<0.01
3 Discussion
Previous studies have shown that several kinds of stresses can induce the adverse alterations of development and reproduction in planarians[3-7]. Our data in the current work further suggest that exposure to heatshock can also cause the defects of development in planarians. Heatshock treatment for different time intervals caused the significant decrease of body weight compared with those in control planarians(Fig 1). Similarly， heatshock treatment for different time intervals resulted in the significant decrease of body length compared with those in control planarians(Fig 2). These data demonstrated that the deficit in development will be formed in planarians exposed to severe heatshock. That is， both the heatshock and the coldshock will exert adverse effects on the development of planarians. Previous study has also demonstrated that the induction of stress protein HSP60 could be detected in planarians after 24h treatment at 27 ℃[4]， implying the severe stress response may be formed in heatshock treated planarians.
Our data further demonstrated that exposure to the severe heatshock will influence the accumulation of lipid and glycogen. We mainly examined the alterations of lipid and glycogen in parenchymal cells of planarians. The reason is that， in planarians， parenchymal cells are located between the epidermal and intestinal cells， and these parenchymal cells are primarily composed of neoblasts， which will account for approximately 30% of the total planarians cells[12]. Treatment with the stress of heatshock inhibited the lipid accumulation in parenchymal cells of planarians as reflected by the sharp decrease of lipid droplet number and reduction of relative size of lipid droplets(Fig 3). In addition， treatment with the stress of heatshock also suppressed the glycogen accumulation in parenchymal cells of planarians as revealed by the decrease of relative intensities of labeled signals for glycogen(Fig 4). Considering the fact that the lipid and glycogen are main sources for energy supply in animals， our data strongly imply that the functions of parenchymal cells may be impaired， and the reduction of lipid and glycogen accumulation may be at least one of the main reasons for the induction of developmental defects in heatshock treated planarians.
Our data further provide the evidence to support such a notion that the accumulation of lipid and glycogen in parenchymal cells is essential for the planarian development. As revealed by the linear regression analysis， the body weight was significantly correlated with the relative size of lipid droplet and the relative intensity of glycogen signal in heatshock treated planarians(Tab 1). Moreover， the body length was also significantly correlated with the relative size of lipid droplet and the relative intensity of glycogen signal in heatshock treated planarians(Tab 2). Therefore， heatshock treatment may influence the planarian development by largely altering the accumulation of lipid and(or) glycogen in parenchymal cells.
Previous study has further suggested that the autophagy plays an essential role in the remodeling of the organism that occurs during regeneration， providing the necessary energy and building blocks to the neoblasts for cell proliferation and differentiation[7]. In addition， evidence have been raised that the accumulation of lipid and glycogen reserves in the parenchymal and gastrodermal cells precedes the aggregation of undifferentiated mitotically active neoblasts beneath the wound after regeneration[13]. Our study strongly imply that the autophagy may be involved in the regulation of both the accumulation of lipid and glycogen in parenchymal cells and the aggregation of undifferentiated mitotically active neoblasts during regeneration in planarians.
Acknowledgements This work was supported by the grants from the National Natural Science Foundation of China(No. 30870810) and the Program for New Century Excellent Talents in University.
参考文献
[1] SNCHEZALVARADO A， TSONIS P A. Bridging the regeneration gap: genetic insights from perse animal models[J]. Nat Rev Genet， 2006，7:873884.
[2] ZHANG Y F， YE B P， WANG， D Y. Molecular actions guiding neural regeneration in planarian[J]. Neurosci Bull， 2008，24:329337.
[3] LI M. Effects of nonionic and ionic surfactants on survival， oxidative stress， and cholinesterase activity of planarian[J]. Chemosphere， 2008，70:17961803.
[4] GUECHEVA T N， ERDTMANN B， BENFATO M S， et al. Stress protein response and catalase activity in freshwater planarian Dugesia(Girardia) schubarti exposed to copper[J]. Ecotoxicol Environ Safety， 2003，56:351357.
[5] KALAFATIC' M， KOVACEVIC' G， FRANJEVIC' D. Resistance of two planarian species to UVirradiation[J]. Folia Biol， 2006，54:103108.
[6] HAMANA K， HAMANA H， SHINOZAWA T. Alterations in polyamine levels of nematode， earthworm， leech and planarian during regeneration， temperature and osmotic stresses[J]. Comp Biochem Physiol B Biochem Mol Biol， 1995，111:9197.
[7] GONZALEZESTEVEZ C， FELIX D A， ABOOBAKER A A， et al. Gtdap1 promotes autophagy and is required for planarian remodeling during regeneration and starvation[J]. Proc Natl Acad Sci USA， 2007，104:1337313378.
[8] AGNES， BRNDSTED H V. Influence of temperature on rate of regeneration in the timegraded regeneration field in planarians[J]. J Embryol Exp Morph， 1961，9:159166.
[9] VOWINCKEL C. The role of illumination and temperature in the control of sexual reproduction in the planarian Dugesia tigrina (Girard)[J]. Biol Bull， 1970， 138:7787.
[10] ACKERMAN G A. A modification of the sudan black B technique for the possible cytochemical demonstration of masked lipids[J]. Science， 1952，115:629631.
[11] HOLMES J H G， ASHMORE C R， ROBINSON D W. Effects of stress on cattle with hereditary muscular hypertrophy[J]. J Anim Sci， 1973， 36∶684694.
[12] KOBAYASHI K， HASHIGUCHI T， ICHIKAWA T， et al. Neoblastenriched fraction rescues eye formation in eyedefective planarian ‘menashi Dugesia ryukyuensis [J]. Dev Growth Differ， 2008，50:689696.
[13] BOWEN I D， DENHOLLANDER J E， LEWIS G H. Cell death and acid phosphatase activity in the regenerating planarians Polycelis tenuis Iijima[J]. Differentiation， 1982，21:160167.