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Changes in iTRAQ-based proteomic profiling of the cladoceran Daphnia magna exposed to microcystin-producing (MP) and microcystin-free (MF) Microcystis aeruginosa Kai Lyu, Qingguo Meng, Xuexia Zhu, Daoxin Dai, Lu Zhang, Yuan Huang, and Zhou Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00101 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 17, 2016

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Environmental Science & Technology

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Changes in iTRAQ-based proteomic profiling of the cladoceran Daphnia magna

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exposed to microcystin-producing (MP) and microcystin-free (MF) Microcystis

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aeruginosa

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Kai Lyu, Qingguo Meng, Xuexia Zhu, Daoxin Dai, Lu Zhang, Yuan Huang, Zhou

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Yang*

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Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological

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Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China

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Address Correspondence to

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*Zhou Yang,

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Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological

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Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China

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E-mail: [email protected]

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Tel: +86-25-85891671

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Fax: +86-25-85891671

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1

ACS Paragon Plus Environment


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ABSTRACT: Global warming and increased nutrient fluxes cause cyanobacterial

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blooms in freshwater ecosystems. These phenomena have increased the concern for

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human health and ecosystem services. The mass occurrences of toxic cyanobacteria

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strongly affect freshwater zooplankton communities, especially the unselective filter

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feeder Daphnia. However, the molecular mechanisms of cyanobacterial toxicity

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remain poorly understood. This study is the first to combine the established body

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growth rate (BGR), which is an indicator of life-history fitness, with differential

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peptide labeling (iTRAQ)-based proteomics in Daphnia magna influenced by

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microcystin-producing (MP) and microcystin-free (MF) Microcystis aeruginosa. A

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significant decrease in BGR was detected when D. magna was exposed to MP or MF

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M. aeruginosa. Conducting iTRAQ proteomic analysis, we successfully identified and

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quantified 211 proteins with significant changes in expression. A cluster of

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orthologous groups revealed that M. aeruginosa-affected differential proteins were

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strongly associated with lipid, carbohydrate, amino acid, and energy metabolism.

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There parameters could potentially explain the reduced fitness based on the cost of

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substance metabolism.

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Table of Contents Art

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INTRODUCTION

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With global warming and remarkable increase in nutrient fluxes, such as nitrates and

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phosphates from agricultural run-off, sewage treatment plants, and other

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anthropogenic sources, the number of cyanobacteria, including toxin-producing taxa,

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has possibly increased; thus, these organisms may pose threats to human and

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environmental health (reviewed by O’Neil, et al. 1). Cyanobacterial blooms can elicit

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detrimental effects on freshwater ecology, economy 2, and human health by altering

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the trophic structure and functionality 3, deoxygenating water columns to cause

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aquatic animal mortalities, and reducing water quality. As a toxin-producing

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cyanobacterium, Microcystis is a common bloom-forming genus. This genus is

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composed of microcystin-producing (MP) and microcystin-free (MF) strains, which

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are distinguished by a 55-kb microcystin synthetase gene cluster 4. Toxigenic (i.e. MP)

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and nontoxigenic (i.e. MF) species are involved in seasonal successions of bloom

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formation and bloom outbreak that vary among lakes 5. The concentration of the

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second metabolic product microcystins in the environment varies from traces amounts

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to 1800 µg L-1 or even higher immediately after the collapse of a highly toxic bloom 6.

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Thus far, more than 100 different microcystin congeners have been identified; these

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congeners mostly result from substitutions of the variable l-amino acids 7.

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Microcystin-LR is the most common and potent variant, followed by microcystin-RR

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and microcystin-YR. On the basis of the LD50 after microcysitns are intraperitoneally

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injected in mice, microcystin-LR with an LD50 value of 50 µg kg−1 is more toxic than 4


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microcystin-RR 8 at 500-800 µg kg−1 and microcystin-YR 8 at 150-200 µg kg−1. Daphnia is a key species in freshwater ecosystems 9. In cyanobacterial blooms,

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the transfer of energy from phytoplankton to Daphnia becomes inefficient because

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cyanobacteria interfere with filter feeding 10, 11, provide inadequate nutrition 12, 13, and

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produce toxic secondary metabolites 14. These factors can contribute to the reduced

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growth, inhibited reproduction, and Daphnia population decline; as a consequence,

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this phenomenon decreases the efficiency of energy transfer from primary producers

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to higher trophic levels 15. Moreover, microcystins may accumulate in primary

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consumers, such as Daphnia; thus, these toxins possibly transfer to higher levels of

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the aquatic food web and may reach humans 16.

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Molecular approaches in ecotoxicology have greatly contributed to the

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mechanistic understanding of the effects of aquatic pollutants on organisms. These

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approaches include high-throughput omics technologies 17. Holistic approaches have

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also been performed with the available transcriptome sequences to reveal the mRNA

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profile of Daphnia in response to Microcystis. Asselman et al. 18 identified four

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Microcystis-affected networks, namely, ribosome, oxidative phosphorylation,

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mitochondrial dysfunction, and protein export networks in Daphnia. The substance

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transportation pathway and energy production have been proposed as adaptive

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mechanisms that provide Daphnia with resistance to toxic cyanobacteria 19, 20.

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Asselman et al.21 further demonstrated that the poor nutritional quality among

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cyanobacterial species can be a potential primary cause of cyanobacterial effects on 5



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Daphnia. These findings are consistent with those described in previous studies 11, 22,

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23

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cyanobacterial toxins are primary inducers of cyanobacterial toxicity. The underlying

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mechanisms of cyanobacterial effects on Daphnia have not been extensively

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addressed because mRNA levels sometimes may be inaccurate predictors of protein

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abundance 24-26. Meanwhile, proteins are the major determinants of biological

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functions in the actual phenotype of an organism; information on the functional

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protein populations should be obtained to understand the relationship between an

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organism and its environment. This understanding can be achieved by analyzing the

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proteome of exposed animals through proteomic technologies. Furthermore,

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molecular modes of toxic action in panoramic form can be potentially improved by

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the complementation of transcriptomics and proteomics.

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at the population level. However, this evidence does not support the hypothesis that

Proteomics is defined as the simultaneous analysis and quantification of cellular

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or extracellular protein abundance. Compared with transcriptomics, proteomics can

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provide functional mechanistic information because mRNA is a disposable message,

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and a limited amount of mRNA is translated into protein. Proteomic techniques can

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detect changes in protein activities determined as posttranslational modifications.

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Despite its advantages, proteomics faces several challenges in ecotoxicology. For

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example, proteomic data are largely dependent on the scale of protein databases;

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inadequate genome annotation lowers the significance of proteomic results 27. The

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direct linking of protein expression profiles to phenotypic and population-level effects 6


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are not easily built, and this drawback also limits transcriptomic analysis. iTRAQ labeling is an efficient quantitative proteomic technology that uses a

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family of isobaric isotope tags to label tryptic peptides from differential protein

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samples; this process is followed by liquid chromatography-tandem mass

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spectrometry (LC-MS/MS) analysis to identify and quantify proteins. The

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LC-MS/MS-based proteome profiling in Daphnia has demonstrated that

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high-throughput proteomics based on the Daphnia genome database is feasible 28.

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Mass spectrometry-based proteomics can be applied to investigate the cyanobacterial

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effects on a Daphnia model system and to elucidate the phenotypic role of functional

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proteins in tolerating toxic cyanobacteria in diet.

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Schwarzenberger et al. 29 reported that the increased proteases activity in

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Daphnia gut facilitated the capacity for protein digestion in the presence of dietary

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protease inhibitors, as a first attempt to explore the response mechanism to

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cyanobacteria at a protein level. The present study aimed to analyze the changes in the

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proteomic profile of Daphnia magna challenged by MP and MF M. aeruginosa. D.

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magna is the most common freshwater invertebrate model species used extensively to

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investigate ecotoxicity 30 because of its relatively high sensitivity to toxicants, rapid

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reproduction, and short lifespan. We examined the effects of dietary exposure to live

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M. aeruginosa cells instead of the effects of individually purified cyanobacterial

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toxins on the basis of previous findings 18. Moreover, studies on the response of

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Microcystis to feeding provide a realistic understanding of its impact on the 7



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environment. Body growth rate (BGR) was utilized as an agent of life-history fitness

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in the presence of MP and MF Microcystis. The animals were examined in terms of

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BGR and used for proteomic analysis. We determined the differentially expressed

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proteins in a pairwise manner because of different food sources. Subsequently, we

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then calculated the significantly affected cluster of orthologous groups (COG) of

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proteins. This study is the first to implement iTRAQ in LC-MS/MS-based proteomics

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to identify cyanobacterium-responsive proteins that mediate life-history changes in

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Daphnia, which can also verify the previous transcriptomic results 18. Furthermore,

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this study aimed to demonstrate how researchers can continue to ecologically improve

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D. magna genome annotation 31, which is characterized by a very high number of

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hypothetical proteins. Some hypothetical proteins may be significantly linked to

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environment-responsive trait variation, but their biological functions have not been

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confirmed in a Daphnia model. When a function of a hypothetical gene/protein is

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identified or when its link to a specific environmental variable (e.g., Microcystis

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exposure considered in this study) is established in the model organism (e.g.,

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Daphnia), the homologs of this gene/-protein in other species can be extensively

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analyzed for their relevance to similar environmental gradients.

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MATERIALS AND METHODS

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Animals and algal cultivation. A clonal culture of D. magna was provided by

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the State Key Laboratory of Pollution Control and Resource Reuse, Nanjing

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University (Nanjing, China) and has been maintained in the laboratory for more than 8


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ten years 14, 20. D. magna was cultured in clean tap water 19 and fed with Scenedesmus

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obliquus (FACHB-416; 1.5 mg C L−1) at 25 °C under fluorescent light at 40 mmol

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photons m−2 s−1 in a light-dark period of 14 h:10 h. The medium was gently aerated

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with filtered air for 24 h before use, and the animals were transferred to a new

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medium twice a week. Brood stocks were maintained at 50 females in 4 L.

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The high-quality alga S. obliquus, the MP M. aeruginosa strain PCC7806, and

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the MF M. aeruginosa strain FACHB-469 were obtained from the Freshwater Algae

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Culture Collection of the Institute of Hydrobiology at the Chinese Academy of

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Sciences. Both M. aeruginosa strains grow as single cells (5-6 µm diameter spheres)

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or paired cells and were chosen to prevent mechanical interference from large

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colonies that might influence Daphnia fitness. We also conducted a pilot experiment

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and confirmed through high-performance liquid chromatography (HPLC) 32 that M.

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aeruginosa PCC7806 produces two types of microcystin, namely, MC-LR and

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MC-RR, with 3.6 pg per cell. Three algal strains were stored in 1 L flasks, which

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contained 400 mL of BG-11 medium 33. The cultures were grown semi-continuously

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at 25 °C under fluorescent light at 40 µmol photons m−2 s−1 supplied in a 14 h:10 h

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light: dark cycle. The strains were harvested in the exponential stage and fed to

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Daphnia. Diet algal densities were determined with a blood cell counting chamber

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under an inverted Nikon microscope at a magnification of 400× and converted to the

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total organic carbon based on a previously developed calibration curve 20.

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Experimental design. The test animals were obtained from the third clutch of D. 9



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magna mothers. These animals were released within 12 h. Afterward, 20 neonates

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from each beaker were transferred to 1000 mL of tap water. These neonates were

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exposed to three different food treatments for 15 days at a total food concentration of

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1.5 mg C L−1: (1) 100% S. obliquus (hereafter labeled as SO); (2) a mixture of 50%

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MP M. aeruginosa + 50% S. obliquus (by carbon content; labeled as MP); and (3) a

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mixture of 50% MF M. aeruginosa + 50% S. obliquus (by carbon content; labeled as

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MF). Food and medium were replaced daily. The three food treatments comprised a

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total of nine biological replicates (three replicates per treatment) in a constant

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photoperiod of 14 h:10 h (light: dark) cycle and at a constant temperature of 25 °C.

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During the experiment, the pH of the medium was monitored at regular intervals and

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stabilized at 7.8±0.2. The BGR of D. magna was determined from the body dry

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weight of a subsample of the animals at the beginning and at the end of the

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experiment (15th day) according to the equation described in a previous study 34. Half

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of the D. magna individuals for BGR determination were quick-frozen after the

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measurements. These individuals were subjected to protein extraction, followed by

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proteomic analyses.

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Measurement of microcystin levels in exposed animals. The remaining D.

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magna individuals were used for microcystin measurement by HPLC 32 with some

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modification. In brief, D. magna were ground in 5 mL of 100% methanol, stirred, and

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left overnight to enable the complete extraction of the microcystins. The methanol

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extract was centrifuged at 9300 × g for 10 min. The supernatant was applied to a 10


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conditioned SPE cartridge (SepPak C18, Waters). The cartridge was first washed with

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5 mL methanol followed by 5 mL distilled water. Impurities were eluted with 2 mL

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methanol and MCs were eluted with 2 mL 80% (v/v) methanol. The eluate was

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analyzed by an Agilent HPLC 1100 system equipped with an ODS (Cosmosil

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5C18-AR, column 250 mm × 4.6 mm, Japan) kept at 40 °C. The mobile phases were

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composed of Milli-Q water containing 0.05% (v/v) trifluoracetic acid and

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HPLC-quality methanol, which were blended at a rate of 45:55 over 25 min. The flow

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rate was 1 mL min-1. The eluent was passed through a variable wavelength detector

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(VWD) operated at 238 nm and calculated against a standard curve with MC-LR and

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MC-RR (Express, China).

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Protein preparation. For each biological replicate of the food experiment, the

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quick-frozen animals from the three different food treatments were transferred into the

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lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5, 1mM

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PMSF, 2mM EDTA) and sonicated in ice. The proteins were reduced with 10 mM

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DTT (final concentration) at 56 °C for 1 h and alkylated by 55 mM IAM (final

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concentration) in the darkroom for 1 h. The reduced and alkylated protein mixtures

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were precipitated by adding 4× volume of chilled acetone at -20 °C overnight. After

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centrifugation at 4 °C, 30,000 × g, the pellet was dissolved in 0.5 M TEAB

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(Applied Biosystems, Milan, Italy) and sonicated in ice. After centrifugation at 30,000

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× g at 4°C, an aliquot of the supernatant was taken to determine the protein

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concentration with a 2-D Quant Kit (GE Healthcare). The proteins in the supernatant 11



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were kept at -80°C for further analysis. iTRAQ labeling and SCX fractionation. The total protein (100 µg) was taken

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from each sample solution, and the protein was digested with Trypsin Gold (Promega,

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Madison, WI, USA) with a protein: trypsin = ratio of 30:1 at 37 °C for 16 hours. After

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trypsin digestion, peptides were dried by vacuum centrifugation, reconstituted in 0.5

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M TEAB, and processed according to the manufacturer’ s protocol for the 8-plex

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iTRAQ reagent (Applied Biosystems). More details of the iTRAQ labeling and SCX

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fractionation are provided in Supporting Information.

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LC-ESI-MS/MS analysis based on Triple TOF 5600. Each fraction was

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re-suspended in buffer A (5% ACN, 0.1%FA) and centrifuged at 20,000×g for 10 min;

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the average final peptide concentration was approximately 0.5 µg µl-1. Subsequently,

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10 µl of the supernatant was loaded in a LC-20AD nanoHPLC appartus (Shimadzu,

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Kyoto, Japan) by the autosampler onto a 2 cm C18 trap column. More details of the

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LC-ESI-MS/MS analysis are provided in Supporting Information.

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Database searches and analysis of identified proteins. Raw data files were

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acquired from the Orbitrap and converted into MGF files by Proteome Discoverer 1.2

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(PD 1.2, Thermo). These MGF files were searched. Protein identification was

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performed by the Mascot search engine (Matrix Science, UK). The search parameters

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included: Gln->pyro-Glu (N-term Q), Oxidation (M), and Deamidated (NQ) as the

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potential variable modifications, and Carbamidomethyl (C), iTRAQ8plex (N-term),

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and iTRAQ8plex (K) as the fixed modifications. The charge states of peptides were 12


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set to +2 and +3. iTRAQ data from three biological replicates were analyzed by the MASCOT

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2.3.02 software before protein identification was performed with Daphnia_nr which

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contained 30455 sequences

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(ftp://ftp.ensemblgenomes.org/pub/release-22/metazoa/fasta/daphnia_pulex/pep/;

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http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi? id=35525). To reduce

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the probability of false peptide identification, only peptides with significance scores

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(≥20) at the 99% confidence interval and a Mascot probability analysis greater than

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“identity” were counted as identified. The confident identification of each protein

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involved at least one unique peptide. For protein quantization, a protein must contain

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at least two unique peptides. The quantitative protein ratios were weighted and

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normalized by the median ratio in Mascot. We only used ratios with P 50%) was observed in MF and MP compared with SO

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(one-way ANOVA, F1,8: 42.107, P < 0.05; Fig. 1). We detected 67 ng of total

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microcystins per gram of D. magna after the 15-day exposure to MP M. aeruginosa.

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Proteomics in Daphnia. The MS/MS spectra were processed with the Mascot

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software. The iTRAQ analysis of D. magna global proteome revealed 7,511 peptides

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in the database (30,455 sequences) and generated 2,232 protein hits in Mascot. Fig. 2

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illustrates the cellular component determined through the GO analysis of the peptides.

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By performing GO analyses, we can categorize the differential proteins into several

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biological process categories, namely, cellular (16.38%), metabolic (14.38%), and

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single-organism (10.61%). We can also classify the proteins according to two

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regulatory stages (Fig. 3), namely, biological regulation (6.56%) and the regulation of

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biological process (5.77%). The major molecular functions of the proteins as obtained

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by GO analysis (Fig. 4) were binding (41.46%) and catalytic activity (41.99%). 14


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We assumed a 1.2-fold increase or decrease in protein expression as a benchmark

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of a significant physiological change. Two pairwise determinations were classified

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into MP/SO and MF/SO and presented in this study.

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A. MP/SO pairwise analysis

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iTRAQ analysis revealed that 94 proteins, including 24 upregulated proteins

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(Supporting Information, Table S1) and 70 downregulated proteins, were quantified

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and showed differential expression after D. magna were exposed to MP M.

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aeruginosa (Supporting Information, Table S2). Among the 24 upregulated proteins,

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10 proteins were involved in metabolism; these proteins included succinyl-CoA

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synthetase for energy production, glutamine-oxaloacetic transaminase for amino acid

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metabolism, protoporphyrinogen oxidase for coenzyme metabolism, and acetyl-CoA

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acetyltransferase for lipid metabolism. Furthermore, 7 proteins were classified as

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hypothetical. Meanwhile, 6 proteins were included in the category of cellular

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processes and signaling, including peroxiredoxin, glutathione S-transferase, and

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tubulin. Only 1 protein was associated with information storage and processing.

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Among the 70 downregulated proteins (Supporting Information, Table S2), 16

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were grouped under information storage and processing. Of these 16 proteins, 11 were

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ribosomal proteins with various subunits. Among the 70 downregulated proteins, 6

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proteins were associated with cellular processing and signaling, such as ABC

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transporter with a 0.66-fold change. Furthermore, 20 metabolism-related proteins

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were identified, including xanthine dehydrogenase for nucleotide metabolism, 15



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pyruvate dehydrogenase for energy production, and dihydropteridine reductase and

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aminopeptidase N for amino acid transport and metabolism. Only 26 of the

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downregulated proteins remained unknown after functional classification was

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performed.

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B. MF/SO pairwise analysis

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A total of 117 proteins were differentially expressed after D. magna were

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exposed to MF M. aeruginosa, including 50 upregulated proteins (Supporting

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Information, Table S3) and 67 downregulated proteins (Supporting Information, Table

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S4). Among the upregulated proteins, 11 proteins were involved in information

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storage and processing, including 7 ribosomal proteins with various subunits.

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Meanwhile, 7 upregulated proteins, such as peroxiredoxin with a 1.577-fold change

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and calponin with a 1.338-fold change, were associated with cellular processing and

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signaling. Furthermore, 9 upregulated proteins were implicated in metabolism and

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transportation, such as glycogen synthase and phosphomannomutase for carbohydrate

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metabolism and 4-aminobutyrate aminotransferase for amino acid metabolism.

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Alpha-spectrin, cuticle proteins, hemoglobin, and 23 other proteins were classified in

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a group without available COG IDs.

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Among the 67 downregulated proteins (Supporting Information, Table S4), the

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translation initiation factor-6 and an RNA-binding protein, were associated with

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information storage and processing; 10 were grouped within cellular processing and

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signaling; 33 were implicated in metabolism and transportation; and 29 were involved 16


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in lipid metabolism, energy production, carbohydrate metabolism, and amino acid

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metabolism. Although 21 proteins did not have an available COG ID, some of these

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proteins exhibited substantially reduced expression levels, such as the hypothetical

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proteins with accession numbers EFX61611, EFX72825, EFX79807, and EFX64299.

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DISCUSSION

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Anthropogenic eutrophication and global warming have altered the composition

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of phytoplankton communities into cyanobacterial populations 1, such as Microcystis.

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Some aquatic herbivores, such as copepods, facilitate cyanobacterial blooms because

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of their selective grazing on non-cyanobacterial prey 35. By contrast, Daphnia species

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are large generalist grazers that inevitably suffer from the harmful effects of

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Microcystis; as such, Daphnia species experience individual growth inhibition 36.

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Given that the BGR of Daphnia is highly correlated with fitness estimates 19, 37-38,

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we demonstrated that chronic dietary exposure to MP and MF Microcystis

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significantly affected the growth of individual Daphnia, as indicated by the reduced

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BGR (Fig. 1). The negative effects of harmful Microcystis on Daphnia can be

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attributed to the toxic effects of MCs 36, the low nutritional value of these cells 13, or

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the mechanical interference in the filtering structures of cladocerans caused by the

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colonial or thick filament morphologies of cyanobacteria 9. The Microcystis strains

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used in the present study primarily consisted of single cells; therefore, the observed

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fitness decrease after Daphnia species were exposed to Microcystis can be attributed

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to toxic substances or poor food quality rather than mechanical obstruction. 17



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This study was designed to explain the life-history changes of the important

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zooplankton Daphnia at the protein level in response to Microcystis, including MP

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and MF strains. Given the advances in analytical techniques, proteomic analysis has

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become a new frontier in molecular biology; this technique can be applied to

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determine the differences or changes in protein expression patterns of organs, cells, or

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subcellular compartments to a reasonably high level of coverage. Over the past few

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years, 2D differential in-gel electrophoresis (DIGE) is a quantitative proteomic

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method that has been successfully applied in Daphnia research which focused on

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predation risk 39-40, protein acetylation 41 and exposure to copper and paraquat 42. As

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an approach for quantifying changes with higher-throughput than DIGE, iTRAQ

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profiling is an emerging and useful technique to quantify changes in global-scale

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proteins (especially for less-abundant proteins) among individuals responsive to

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various environmental stressors 43-45. To the best of our knowledge, the present study

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is the first to use an iTRAQ approach to identify and quantify proteomes of

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zooplankton mobilized against environmental pollutants, even though iTRAQ was

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previously used to study anti-predation response in Daphnia 34. To acquire valuable

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evidence supporting the global-scale proteomic changes in Daphnia in response to

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cyanobacterial effects, we discussed our results in two pairwise comparisons, namely,

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MP/SO and MF/SO.

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Interpretation of differentially expressed proteins in MP/SO. Among the

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upregulated proteins associated with cellular processes and signaling (Supporting 18


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Information, Table S1), peroxiredoxin and glutathione S-transferase (GST) are

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antioxidant enzymes with important roles in protecting organisms against the toxicity

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of reactive oxygen species. Oxidative stress is a toxicological consequence of MC

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exposure in different organisms 46. Peroxiredoxin catalyzes the reduction of hydrogen

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peroxide, organic hydroperoxides, and peroxynitrites to less reactive products; thus,

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this enzyme is implicated in the first line of defense against oxidative stress and key

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apoptosis events 47, which are potentially caused by MCs 48. As a phase-II

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detoxification enzyme, GST has been identified as upregulated by MC exposure; this

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finding is consistent with previous studies, which revealed that an increase in GST

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gene expression is induced by the oxidative stress caused by the toxin 18 and the

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conjugation of MC with glutathione (GSH) during detoxification 49. Sadler and von

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Elert 50 investigated whether a cyanobacterial diet of the MP Microcystis causes the

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biotransformation of MC conjugation products in a Daphnia clone in vivo; their group

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found that GSTs in Daphnia are involved in oxidative stress response rather than in

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the specific biotransformation of MCs conjugated with GSH. The increased GST and

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peroxiredoxin in this study corresponded to the enhanced antioxidation ability of

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Daphnia in response to cyanobacterial effects.

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Serine/threonine protein kinase (STPK) is a sensor of the unfolded protein

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response pathway; this enzyme plays a crucial role in the homeostatic regulation of

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protein folding and in stress response to cope with an increased number of unfolded

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proteins 51. Therefore, exposure to MP Microcystis possibly interfered with the normal 19



375

modification of protein because higher STPK protein expression was induced by

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Microcystis. However, transcriptome profiling revealed that the STPK gene

377

expression was inhibited in Daphnia fed with MP Microcystis18. This contradictory

378

result may be attributed to the increased MC content (80 ng > 67 ng in our study) that

379

accumulated in Daphnia. Alternatively, complicated and varied post-transcriptional

380

mechanisms involved in turning mRNA into protein may cause the absence of

381

positive correlation in protein abundance and mRNA expression levels 52. Therefore,

382

we cannot exclude the possibility that the down-regulated STPK gene expression is a

383

result of feedback control by sufficient levels of the STPK protein in the cell.

384

Ribosomal proteins were mostly identified in the information storage and processing

385

(Supporting Information, Table S2). These downregulated proteins were related to the

386

inhibited protein synthesis in Daphnia exposed to MP Microcystis; this finding has

387

been confirmed by transcriptomic analysis 18.

388

One of the cytotoxic events triggered by MC that was first to be described is

389

cytoskeleton disruption 53, which is a frequent reaction to toxic substances. The

390

microtubule dynamics in cells partially relies on tubulin post-translational

391

modifications, such as phosphorylation, which is controlled by the balance between

392

protein phosphatases and kinases. MCs are strong inhibitors of protein phosphatase 1

393

and 2A, which eventually cause the hyperphosphorylation of cytoskeletal proteins,

394

such as tubulin 54. Tubulin maintains the cellular architecture, internal organization,

395

cell shape, motility, cell division, and other processes 55. MCs may disrupt the 20


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Page 21 of 41


396

cytoskeletal structure by directly binding to tubulin cysteine residues after toxins enter

397

cells. Ding et al. 56 reported the morphological changes in microtubules from evenly

398

distributed structures to dense aggregations after Daphnia was treated with

399

Microsystis extract. Our study revealed that the upregulated tubulin protein was

400

stimulated by Microsystis, thereby further suggesting that MP Microsystis affected the

401

quantitative changes in microtubules.

402

Some upregulated proteins, such as succinyl-CoA synthetase (SCS), are involved

403

in metabolism to enable Daphnia to cope with MP Microcystis. SCS is a

404

hydrogenosomal enzyme that catalyzes the formation of ATP via substrate-level

405

phosphorylation 57. The observed upregulation of SCS protein is related to cushion

406

energy deficiency in Daphnia exposed to MP Microcystis. Nevertheless, the number

407

of downregulated metabolic proteins was more than that of the upregulated proteins.

408

These downregulated proteins were involved in the metabolism of carbohydrates,

409

amino acids, lipids, and energy. Ribulose-5-phosphate 4-epimerase (RPE) is an

410

enzyme that catalyzes the interconversion of ribulose 5-phosphate and xylulose

411

5-phosphate in the oxidative phase of the pentose phosphate pathway (PPP) 58. As the

412

main product of PPP, nicotinamide adenine dinucleotide phosphate is essential for

413

lipid production. Therefore, the reduced RPE, which is induced by MP Microcystis,

414

may result in carbohydrate metabolism disturbances, lipid production inhibition, and

415

enhanced energy deficiency. Pyruvate dehydrogenase (PD), which synthesizes

416

high-energy compounds, such as ATP 59, is also required for glycolysis. We found that 21



417

the presence of MP Microcystis downregulated PD protein expression; this finding

418

confirmed the potential energy deficiency.

419

Considering that MCs constitute a family of potent cyclic heptapeptide toxins 46,

420

we found that the degradation of heptapeptides that depends on amino acid

421

metabolism is a direct approach to buffer the cytotoxicity of MCs in exposed

422

individuals. Trypsin-like serine proteases are remarkably affected by MP Microcystis

423

with an approximately 0.3-fold change. A trypsin-like serine protease cleaves peptide

424

chains mainly at the carboxyl side of the amino acid lysine or arginine; this protease is

425

the major compound in the digestive system of amino acids in the Daphnia gut 60.

426

Czarnecki et al. 61 found a Microcystis-induced decrease in the trypsin activity of

427

Daphnia; this result is consistent with that obtained of our study. Aeruginosins are

428

toxins produced by M. aeruginosa 62; these toxins inhibit trypsin-like serine proteases

429

by directly interacting with proteins. A previous study19 suggested that the transport of

430

MCs out of a cell is carrier mediated and involves certain permeases. We also found a

431

decrease in the permease and ATPase components of Daphnia subjected to long-term

432

exposure to MP Microcystis. Therefore, the inhibited transport of MC out of cells and

433

the decreased degradation of MCs could increase the toxin accumulation in cells.

434

Interpretation of differentially expressed proteins in MF /SO. Although

435

studies on the interaction of Daphnia with MP Microcystis have been extensively

436

conducted, research on the interactions between MF Microcystis and Daphnia has

437

been rarely performed probably because of the lack of toxins. However, poor somatic 22


Page 22 of 41

Page 23 of 41


438

growth, survival, and reproduction of Daphnia have been observed when the species

439

is cultured on mixtures containing good green algal food with MF Microcystis63.

440

Nutritional deficiency in some polyunsaturated fatty acids that essential for

441

zooplankton growth may also reduce the food quality of cyanobacteria64. By

442

considering the hypothesis related to fatty acids, Demott and Müller‐Navarra 65 found

443

that the growth rates of three Daphnia species are markedly reduced when these

444

species are fed with a MF cyanobacterium as a sole food source. However, growth is

445

promoted when an emulsion rich in omega-3 fatty acids is mixed with the

446

cyanobacterium. This finding suggests that Synechococcus lacks lipids that are

447

essential for Daphnia growth. Nevertheless, limited molecular evidence explains the

448

serious effects of toxin-lacking cyanobacterium on zooplankton.

449

In the present study, iTRAQ proteomic analysis was applied to investigate

450

potential protein changes in Daphnia exposed to MF Microcystis. Insufficient

451

essential lipids in the toxin-free cyanobacterium were considered as the main factor

452

contributing to the growth reduction of Daphnia. Therefore, the exposure to MF

453

Microcystis may trigger lipid metabolism dysfunction. Our proteomic results verified

454

our prediction that the four lipid-metabolic proteins are downregulated; by contrast,

455

no lipid-metabolic proteins are upregulated in Daphnia exposed to MF Microcystis

456

(Supporting Information, Table S4). Long-chain fatty acid-CoA ligase (also called

457

fatty acid acyl-CoA synthetase) play an essential role in lipid biosynthesis and fatty

458

acid degradation; this enzyme catalyzes the conversion of free fatty acids into fatty 23



459

acyl-CoA esters 66. This reaction is essential for energy storage. The three remaining

460

lipid-metabolic proteins involved in the synthesis and degradation of ketone bodies

461

were also identified as 3-ketoacid CoA transferase, 3-hydroxyacyl-CoA

462

dehydrogenase, and acetyl-CoA acetyltransferase. The ketone body pathway is

463

normally used in mammals as an energy reserve when the citric acid cycle becomes

464

saturated with acetyl-CoA from fatty acid oxidation 67. In invertebrates, the

465

metabolism of ketone bodies has been poorly studied; in insects, the concentration of

466

acetoacetate increases after long periods of starvation, and ketone bodies are used as

467

an energy source for development 68. The low protein expression of 3-ketoacid CoA

468

transferase in Daphnia suggested that ketone bodies might be degraded; this finding

469

indicated a probable pathway that weakens energy production via the citric acid cycle.

470

In addition to lipid metabolic dysfunction-related proteins, the downregulation of

471

7 carbohydrate-metabolic proteins, such as hexokinase and

472

fructose-1,6-bisphosphatase was induced by MF Microcystis. Hexokinase catalyzes

473

the first step in the oxidative metabolism of hexoses via glycolysis.

474

Fructose-1,6-bisphosphatase is an enzyme that converts fructose-1,6-bisphosphate to

475

fructose-6-phosphate via gluconeogenesis. Glycolysis and gluconeogenesis achieve a

476

dynamic balance between the generation and utilization of glucose; these pathways

477

are also involved in energy production. Therefore, the low expression of

478

carbohydrate-metabolic proteins, together with lipid-metabolic proteins, indicated the

479

energy deficiency in Daphnia exposed to MF Microcystis. The proteins directly 24


Page 24 of 41

Page 25 of 41


480

related to energy production exhibited reduced expression (Supporting Information,

481

Table S4). Among these energy-related enzymes, the enzyme lactate dehydrogenase

482

(LDH; converting pyruvate to lactate and vice versa) is involved in the supply or

483

storage of energy from carbohydrates. The decreased LDH activity was also found in

484

Daphnia exposed to MCs 69. These findings are consistent with those observed in our

485

study. However, we cannot assume that MF and MP Microcystis share the same

486

pathway that interrupts energy production in Daphnia because the energy-related

487

NAD-dependent aldehyde dehydrogenases was detected (Supporting Information,

488

Tables S2 and S4).

489

Interpretation of hypothetical proteins identified in pairwise MP/SO and

490

MF/SO. After the publication of the D. pulex genome 31, research on Daphnia has

491

provided new insights into the genome structure, function, and expression associated

492

with the variation in ecologically important traits. The partially annotated genome

493

information of D. magna and D. pulex facilitates the proteomic identification and

494

characterization of proteins by existing databases. However, we found that 36% of the

495

differentially expressed proteins were hypothetical proteins without verified

496

biological functions in vivo. Some of these hypothetical proteins showed significant

497

changes, such as EFX67764 (Supporting Information, Table S2) with 0.313-fold

498

change, EFX77516 (Supporting Information, Table S3) with 5-fold change and

499

EFX64299 (Supporting Information, Table S4) with 0.338-fold change. The

500

gene/protein functions and their relationship with phenotypes can be fully elucidated 25



501

based on the the ecological context of organisms and natural selection. Thus, further

502

studies should be performed to confirm the ecological function of hypothetical

503

proteins. Hypothetical proteins with significant changes can be considered as

504

appropriate first targets.

505

In conclusion, this study described the large-scale effects of Microcystis on the

506

changes in the proteome of D. magna through a high-throughput iTRAQ labeling

507

quantitative technology. With this technology, 94 and 117 differentially expressed

508

proteins were determined after Daphnia was exposed to MP and MF Microcystis,

509

respectively. These proteins were mostly associated with lipid, carbohydrate, amino

510

acid, and energy metabolism. These proteins may correspond to changes in

511

metabolism necessary to adjust the BGR of Daphnia. In addition, hypothetical

512

proteins detected in the model organism Daphnia should also be considered to

513

enhance our understanding of their functionally explicit relationship with phenotypes

514

on the basis of environmental context.

515

ASSOCIATED CONTENT

516

Supporting Information Available

517

Details on iTRAQ labeling, SCX fractionation and LC-ESI-MS/MS analysis.

518

Table S1 describes the upregulated proteins in Daphnia magna fed with 50% S.

519

obliquus + 50% MP M. aeruginosa compared to those fed with 100% S. obliquus.

520

Table S2 describes the downregulated proteins in D. magna fed with 50% S. obliquus

521

+ 50% MP M. aeruginosa compared to those fed with 100% S. obliquus. Table S3 26


Page 26 of 41

Page 27 of 41


522

describes the upregulated proteins in D. magna fed with 50% S. obliquus + 50% MF

523

M. aeruginosa compared to those fed with 100% S. obliquus. Table S4 describes the

524

downregulated proteins in D. magna fed with 50% S. obliquus + 50% MF M.

525

aeruginosa compared to those fed with 100% S. obliquus. This information is

526

available free of charge via the Internet at http://pubs.acs.org/.

527

NOTES

528

The authors declare no competing financial interest.

529

ACKNOWLEDGMENTS

530

We thank the three anonymous reviewers for their helpful comments. This study was

531

supported by the National Basic Research Program of China (2012CB956102), the

532

National Natural Science Foundation of China (31270504), NSFC for Talent Training

533

in Basic Science (J1103507), and the Priority Academic Program Development of

534

Jiangsu Higher Education Institutions.

535

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1342-1349. 35



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Figure Captions

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Figure 1. Changes in body growth rate (BGR) of Daphnia magna fed with different

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food treatments containing either 100% Scenedesmus obliquus (SO), 50% S. obliquus

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+ 50% microcystin-free Microcystis aeruginosa (MF), or 50% S. obliquus + 50%

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microcystin-producing M. aeruginosa (MP). Significant difference (P < 0.05) among

752

treatment levels was detected by one-way ANOVA followed by Duncan’s multiple

753

range test.

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Figure 2. Gene ontology (GO) analysis of the proteins of Daphnia magna in terms of

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cellular component.

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biological process.

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molecular function.

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37



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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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