Background: Marine medaka is among the most popular models of fish species for ecotoxicology and environmental research and proteomic studies are useful tools for understanding the molecular responses of medaka upon exposure to different environmental stressors. The preparation of high-quality protein samples is the key to producing high-quality two-dimensional gel electrophoresis (2-DE) results for proteomic analysis. In recent years, Trizol-based protein extraction has been gaining popularity because of its promising performance in producing high-quality 2-DE as well as the convenience of the method. Methods: Three Trizol-based approaches (Trizol method, Aliquot Trizol method and Trizol method with a commercial clean-up kit) were used to extract proteins from a marine medaka sample and 2-DE profiles were produced. Quality of the 2-DE profiles and effectiveness of the extraction methods were evaluated. For comparison, two common protein extraction methods (lysis buffer method and trichloroacetic acid (TCA)/acetone precipitation extraction) were also applied in parallel to Trizol-based approaches. Results: Any of the three Trizol-based approaches produced a high-quality 2-DE profile of marine medaka compared with both lysis buffer method and TCA/acetone precipitation extraction. In addition, Trizol method with a commercial clean-up kit produced the best 2-DE profile in terms of background clarity, number of spots and resolution of proteins. Conclusions: Trizol-based approaches offered better choices than traditional protein extraction methods for 2-DE analysis of marine medaka. The modified version of Trizol method with a commercial clean-up kit was shown to produce the best 2-DE profile.

Two-dimensional gel electrophoresis (2-DE) is a major element of conventional gel-based proteomics, which resolves complex protein mixtures. Protein extraction with the removal of interfering substances from the sample remains the key to producing high-quality 2-DE profiles. Marine dinoflagellates contain large endogenous amounts of salts, nucleic acids, polysaccharides, phenolic compounds, pigments, and other interfering compounds. These substances are detrimental to the quality of gel images. Protein preparation using TRIzol reagent is a promising method for producing high-quality 2-DE profiles for dinoflagellate samples. In addition to its remarkable performance, the TRIzol method’s several advantages have made it a popular and widely used method in the field of 2-DE sample preparation. Nonetheless, the quality of 2-DE of samples from certain dinoflagellate species is not as high as previously reported when the same TRIzol protocol is applied. Therefore, modifications to the original TRIzol method are required to remove interfering substances from those challenging dinoflagellate samples. In this study, the original TRIzol method and four modified methods, namely the aliquot TRIzol method, re-TRIzol method, TRIzol method with a commercial clean-up kit, and TRIzol method with trichloroacetic acid/acetone precipitation, were compared. Performance of these five methods in terms of protein yield, background signal, and resolution and number of protein spots was investigated on samples from two benthic Prorocentrum species: P. lima and P. hoffmannianum. Our results demonstrated that high-quality 2-DE could be achieved from P. lima samples prepared using both the original TRIzol method and the TRIzol method with a commercial clean-up kit. However, the original TRIzol method failed to produce high-quality 2-DE profiles for P. hoffmannianum samples. Among the four modified TRIzol methods, only the TRIzol method with a commercial clean-up kit could yield substantially improved high-quality 2-DE profiles for P. hoffmannianum samples. This combination of the conventional TRIzol method with a commercial clean-up kit potentially represents a promising protein extraction methodology for obtaining high-quality 2-DE profiles for difficult dinoflagellate samples.

Karenia mikimotoi is one of the most damaging ichthyotoxic dinoflagellate species commonly found in China. However, its growth and ichthyotoxicity responses to salinity changes are still largely unknown. In this study, the growth and ichthyotoxicity of three K. mikimotoi strains, Hong Kong strain KMHK, Japanese strain NIES2411 and New Zealand strain CAWD133, under different salinities (25 to 35 ppt), initial algal densities (5 to 40 thousand cells) and growth phases were investigated. Results indicated that the optimum salinity for all three strains was 30 ppt. The Japanese strain achieved the highest maximum cell densities (cells mL−1) and the New Zealand strain achieved the highest specific growth rate. The Hong Kong and New Zealand strains could not tolerate the low salinity at 25 ppt and the algal cells burst after 3 days of exposure. The average cell widths of all three algal strains in 35 ppt salinity were significantly larger than that in 30 ppt. The acute toxicity test performed on Oncorhynchus mykiss gill cell line RTgill-W1 revealed that the median lethal times for KMHK and NIES2411 were 66.9 and 31.3 min, respectively, and their ichthyotoxicity was significantly affected by algal cell density and growth phase. Nevertheless, CAWD133 did not pose any ichthyotoxicity. The gill cell viability levels at 30 min were reduced from 96 to 61% and 95 to 39% for KMHK and NIES2411, respectively, when the algal cell density increased from 5 × 103 to 4 × 104 algal cells mL−1. Both KMHK and NIES2411 at stationary phase also had higher toxicity than at log phase, with a 27% reduction of gill cell viability, and exerted higher toxicity to the gill cells under extremely low (28 ppt) or high (35 ppt) salinity. These findings demonstrated that the growth–ichthyotoxicity response of Karenia mikimotoi to salinity was not only strain-specific but also depended on its density and growth phase. Study on the effects of salinity on the growth and toxicity of K. mikimotoi is greatly limited. Results from the present study provide valuable insight on the growth and toxicity of different K. mikimotoi strains, which is important in understanding their occurrence of algal bloom and fish-killing action.

Karenia mikimotoi is a well-known harmful algal bloom species. Blooms of this dinoflagellate have become a serious threat to marine life, including fish, shellfish, and zooplanktons and are usually associated with massive fish death. Despite the discovery of several toxins such as gymnocins and gymnodimines in K. mikimotoi, the mechanisms underlying the ichthyotoxicity of this species remain unclear, and molecular studies on this topic have never been reported. The present study investigates the fish-killing mechanisms of K. mikimotoi through comparative proteomic analysis. Marine medaka, a model fish organism, was exposed to K. mikimotoi for a three-part time period (LT25, LT50 and LT90). Proteins extracted from the whole fish were separated by using two-dimensional gel electrophoresis, and differentially expressed proteins were identified with reference to an untreated control. The change in fish proteomes over the time-course of exposure were analyzed. A total of 35 differential protein spots covering 19 different proteins were identified, of which most began to show significant change in expression levels at the earliest stage of intoxication. Among the 19 identified proteins, some are closely related to the oxidative stress responses, energy metabolism, and muscle contraction. We propose that oxidative stress-mediated muscle damage might explain the symptoms developed during the ichthyotoxicity test, such as gasping for breath, loss of balance, and body twitching. Our findings lay the foundations for more in-depth studies of the mechanisms of K. mikimotoi’s ichthyotoxicity.

Some strains of the dinoflagellate species Prorocentrum hoffmannianum show contrasting ability to produce diarrhetic shellfish poisoning (DSP) toxins. We previously compared the okadaic acid (OA) production level between a highly toxic strain (CCMP2804) and a non-toxic strain (CCMP683) of P. hoffmannianum and revealed that the cellular concentration of OA in CCMP2804 would increase significantly under the depletion of phosphate. To understand the molecular mechanisms, here, we compared and analyzed the proteome changes of both strains growing under normal condition and at phosphate depletion using two-dimensional gel electrophoresis (2-DE). There were 41 and 33 differential protein spots observed under normal condition and phosphate depletion, respectively, of which most were upregulated in CCMP2804 and 22 were common to both conditions. Due to the lack of matched peptide mass fingerprints in the database, de novo peptide sequencing was applied to identify the differentially expressed proteins. Of those upregulated spots in CCMP2804, nearly 60% were identified as peridinin-chlorophyll a-binding protein (PCP), an important light-harvesting protein for photosynthesis in dinoflagellates. We postulated that the high expression of PCP encourages the production of DSP toxins by enhancing the yields of raw materials such as acetate, glycolate and glycine. Other possible mechanisms of toxicity related to PCP might be through triggering the transcription of non-ribosomal peptide synthetase/polyketide synthase genes and the transportation of dinophysistoxin-4 from chloroplast to vacuoles.