Trade-off Strategies And Cost Analysis Of Inducible Defenses Of Scenedesmus Obliquus And Differences At The Transcriptome Level

Many organismsat different trophic levels have developed various defensive strategies to avoid the predation by species at higher trophic levels, during the co-evolution with their predators. Inducible defense and constitutive defense are the two major types, based on their timeliness. Scenedesmus spp., a world-wide freshwater phytoplankton, has typical inducible defense. It forms coenobia of 4, 8 or even more cells in response to the infochemicals released by predators (e.g. rotifer,cladoreans). The formation of colonies can effectively reduce the grazing efficiency of zooplankton. According to the defense theory, any types of defense are accompanied by costs. Due to the respectively lower ratios of surface to volume of multi-celled colonies, the package effect impairs the absorption of light and nutrients, and the higher sinking ratio causes higher risk of sedimentation. However, in the previous studies, the direct costs (e.g. the reduction of growth rate) of forming induced colonies have not been detected yet. As an important model for the study of inducible defense theory, the costs of defense in Scenedesmus is an urgent problem to solve. For the previous studies were conducted in high quality environments, the package effects might be denied. In the present study, we explored the presence of direct costs accompanied with the inducible defense in S. obliquus by reducing the availability of environmental resources. In addition, the existence of costs will lead to the trade-offs between defense to predation and environmental disturbances when the organisms are facing multiple environmental selective pressures. To figure out the defensive strategy of S. obliquus, experiments of interspecific competition, exposure to environmental pollutions and long-term repeated exposure to Daphnia infochemicals were conducted.The present study exploredthe differential gene expression at the level of transcriptome by RNA-sequencing. The major results of the present study included 6 aspects as below:1. Trade-offs and direct costs of the inducible defense in S. obliquus under the deficiency of environmental resourcesThe inducible defense of S. obliquus relies on the sufficient availability of environmental resources. The expression of defensive colonies was reduced under the deficiency of environmental resources. Different species of the essential environmental resources have different effects on the colony formation. Light limitation restricted the defensive colony size. The maximum colony size decreased along with the decreasing phosphorous concentrations. The decreased availability of another important element to photoautotrophic organism, nitrogen, did not affect the maximum defensive colony size but prolonged the maintaining time. The changes of defensive colony formation by the insufficient availability of two metallic elements,magnesium and calcium, was similar with phosphorous. Under the deficiency of Mg2+and Ca2+, less colonies will be formed in response to Daphnia infochemicals. The contents of the soluble and bound extracellular and intercellular polysaccharides showed significant increases under the exposure to Daphnia filtrate. The limitation of nitrogen .increased the production of polysaccharides, but the limitation of phosphorous had no significant effect on the contents of polysaccharides. Generally speaking, trade-offs existed between defense and growth. To avoid the costs in cellular growth, the reduced availability of phosphorous, light, Mg2+ and Ca2+ limited the expression of inducible defense. However, nitrogen limitation prolonged the maintenance of defensive colonies. Under the deficiency of phosphorous and Mg2+,direct costs for high growth rate accompanied with the formation of colonies were detected.2. Trade-offs between competitive ability and inducible defense intensityWhen facing both competition pressure and grazing pressure, trade-offs were determined to be existence between the formation of large sinkable colonies (favoring defense) and the maintenance of vulnerable unicells (favoring competition) in S.obliquus. In the presence of Microcystis aeruginosa, Chlorella sp. and Synedra sp.,the strength of inducible defense of S. obliquusdecreased. The growth inhibition of S.obliquus was detected with the expression of defensive colonies which led to the reduction of competitive ability of S. obliquus.A negative relationship between the biomass of co-existing species and the strength of forming defensive colonies was observed. When biomass of competitors was high, high expression of defensive colonies was sacrificed to maintain a higher competitive ability. The present experiments indicate the exist of trade-offs between competition and defense.3. The effects of secondary metabolites of interspecies on the inducible defense of S.obliquusIn the previous chapter, we explored the effects of microcystin (MC-LR),produced by Microcystis and other cyanobacteria, and the exudates of macrophytes on the inducible defense inS. obliquus. Under the exposure of MC-LR, neither the formation of defensive colonies nor the growth of S. obliquus changed significantly.The exudates of four species of macrophytes enhanced the inducible defense of S.obliquus. Additionally, the algal cell multiplication was interfered by the exudates.The results indicated a weaker effect of MC-LR, and a higher effect of macrophytes exudates on S. obliquus. The enhanced colony formation of S. obliquus influenced by macrophytes was at the costs of normal population growth.4. Heavy metal and surfactants interfered the inducible defense inS. obliquusNonlethal concentrations of heavy metals (Zn2+) were figured out to have great inhibition of inducible colony formation in S. obliquus. Elevated temperature enhanced the toxicity of Zn2+ on the defense and growth of S. obliquus. Three kinds of surfactants (sodium dodecyl sulfate, SDS; benzalkonium bromide, BZK; and nonylphenol polyoxyethylene ether, NPE40), at very low environmental concentrations, disturbed the intensity of inducible defense inS. obliquus even with no effect on growth. SDS, the anionic surfactant, increased the proportions of colonies both in the presence and the absence of Daphnia filtrate. The cationic surfactant (BZK)and nonionic surfactant (NPE40) inhibited the formation of colonies in S. obliquus.The effects of the four environmental pollutants on the inducible defense of S.obliquus were concentration related. This chapter indicated the interference of environmental pollutants on the inducible defense of S. obliquus.5. Trade-offs of inducible defense under long-term repeated exposures to Daphnia infochemicals in S. obliquusIn our study, two different patterns of long-term repeated exposures to Daphniainfochemicals were conducted: 1) re-addition of Daphnia filtrate when the defensive colonies disaggregated; 2) re-addition of Daphnia filtrate at the peak of colony formation. The defensive colony formation was reduced during long period exposure to infochemicals in both patterns. In the first pattern,the maximal average colony size exhibited a significant decrease in the 4th round. After 10 rounds,the colony size was about 2.5 cells per colony. In the second pattern,the intensity of inducible defense decreased quickly. The average colony size decreased to 2.5 cells per colony in the 4th round. In the 5th round, the colony size was below 2 cells per colony and the efficiency of inducible defense decreased. The results indicated downward trends of inducible defense in S. obliquusunder the long-term repeated exposures to Daphniainfochemicals, and a significant relationship between the frequency of exposure and the reduction of inducible defense.6. Transcriptome profiling reveals the regulatory mechanism of the inducible defense in S. obliquusThe present study focused on the transcriptome to defensive S. obliquus, and revealed that several pathways were enriched in the process of anti-grazer morph alternations. The inositol phosphate metabolic pathways and plant-pathogen interactions might work as the capture and transmission pathway of Daphnia infochemicals. The up-regulation of photosynthetic carbon fixation increased the production of carbohydrates; and the metabolisms of starch and sucrose as well as metabolism of galactose supplied the raw materials of extracellular polysaccharides.The down-regulated pathways(e.g. biosynthesis of ribosome, antioxidant enzyme systems and biosynthesis of folate) indicated the formation of inducible defense was at the costs of synthesis of proteins, anti-oxidation, cellular division, and other processes.In conclusion, the present study systematically analyzed the direct costs in the formation of inducible defense in S. obliquus, investigated the trade-offs between the defense of predator and other environmental selective pressures, and explored the regulatory mechanism at the level of transcriptome. The results improved the theory of inducible defense in Scenedesmus and had special meaning to the development ofinducible defense theory.