Benthic Cyanobacteria (Oscillatoriaceae) That Produce Microcystin-LR, Isolated from Four Reservoirs in Southern California
Total Page：16
File Type：pdf, Size：1020Kb
ARTICLE IN PRESS WATER RESEARCH 41 (2007) 492– 498 Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/watres Benthic cyanobacteria (Oscillatoriaceae) that produce microcystin-LR, isolated from four reservoirs in southern California George Izaguirrea,Ã, Anne-Dorothee Jungblutb, Brett A. Neilanb aWater Quality Laboratory, 700 Moreno Avenue, Metropolitan Water District of Southern California, La Verne, CA 91750, USA bSchool of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, 2053 New South Wales, Australia article info ABSTRACT Article history: Cyanobacteria that produce the toxin microcystin have been isolated from many parts of Received 9 November 2005 the world. Most of these organisms are planktonic; however, we report on several Received in revised form microcystin-producing benthic filamentous cyanobacterial isolates from four drinking- 3 October 2006 water reservoirs in southern California (USA): Lake Mathews, Lake Skinner, Diamond Valley Accepted 4 October 2006 Lake (DVL), and Lake Perris. Some samples of benthic material from these reservoirs tested Available online 28 November 2006 positive for microcystin by an ELISA tube assay, and all the positive samples had in Keywords: common a green filamentous cyanobacterium 10–15 mm in diameter. Seventeen unialgal Cyanobacteria strains of the organism were isolated and tested positive by ELISA, and 11 cultures of these À1 Cyanotoxins strains were found to contain high concentrations of microcystin-LR (90–432 mgL ). The Lyngbya cultures were analyzed by protein phosphatase inhibition assay (PPIA) and HPLC with Microcystin photodiode array detector (PDA) or liquid chromatography/mass spectrometry (LC/MS). Phormidium Microcystin per unit carbon was determined for six cultures and ranged from 1.15 to 4.15 mgmgÀ1 C. Phylogenetic analysis of four cultures from Lake Skinner and DVL using cyanobacterial-specific PCR and sequencing of the partial 16S rRNA gene suggested the highest similarity to an unidentified cyanobacterium in the oscillatoriales, and to a Phormidium sp. Morphologically, some of the isolates were similar to Oscillatoria, and others resembled Lyngbya. The significance of these organisms lies in the relative scarcity of known toxin producers among freshwater benthic cyanobacteria, and also as a source of cell-bound microcystin in these reservoirs. & 2006 Elsevier Ltd. All rights reserved. 1. Introduction and the occurrence of benthic filamentous cyanobacteria in their water source. Identification of these species has Cyanobacteria that produce the toxin microcystin have been important implications for understanding the ecophysiology isolated from many parts of the world, the majority being of microcystin production and water-quality management. planktonic species (Sivonen and Jones, 1999). The detection of Microcystin is an inhibitor of eukaryotic-type protein large benthic microcystin-producing cyanobacterial popula- phosphatases 1 and 2A, and the target organ is the liver. For À1 tions is rare and to date only a few studies have been microcystins, LD50 levels up to 50 mgkg (i.p. mouse) have reported, such as Mez et al. (1997) and Dasey et al. (2005). been recorded (Botes et al., 1984; Kaya and Watanabe, 1990). Correlations have been made between the poisoning of cattle Microcystin is a cyclic heptapeptide, with a general structure ÃCorresponding author. Tel.: +1 909 392 5127; fax: +1 909 392 5246. E-mail address: [email protected] (G. Izaguirre). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.10.012 ARTICLE IN PRESS WATER RESEARCH 41 (2007) 492– 498 493 of cyclo-(D-alanine-X-D-MeAsp-Z-Adda-D-glutamate-Mdha). The objectives of this study were to determine the source of D-MeAsp is D-b-erythro-methyl-aspartatic acid, and Mdha is microcystin in benthic material from the reservoirs in N-methyldehydroalanine; X and Z are variable L-amino acids. question, to identify the particular toxin variant(s) involved, So far, more than 70 different types of microcystin have been and to relate the toxin producer to other known toxic identified (Sivonen and Jones, 1999) the most common types cyanobacteria. being microcystin-LR, -RR, and -YR, where the variable amino acids (X and Z) are leucine–arginine and tyrosine. Microcystin production can be found in all cyanobacterial 2. Materials and methods orders; however, it is most often found in cyanobacterial blooms consisting of Microcystis, Planktothrix, and Anabaena. 2.1. Isolation of toxin producer The genus Phormidium, however, is known for the production of novel bioactive compounds which have hepatotoxic and Samples of benthic algal growths were collected by SCUBA neurotoxic effects in mice (Baker et al., 2001; Teneva et al., divers from Lake Mathews, Lake Skinner, DVL, and Lake Perris 2005). Moreover, the production of cyanobacterial alkaloid as part of routine sampling for taste-and-odor monitoring. toxins such as saxitoxin (Teneva et al., 2005) and homo- Samples of algal material were collected in 125 ml plastic anatoxin-a (Lilleheil et al., 1996) has been reported for the bottles at various standard sampling locations, usually at a genus Phormidium. depth of 6 m, and transported to the laboratory the same day. The Metropolitan Water District of southern California A map and description of some of the sites in Lake Mathews (MWDSC) supplies drinking water to about 18 million people can be found in McGuire et al. (1984). For a description of DVL in six counties in the coastal plain of southern California and a discussion of problem cyanobacterial events in that (USA). MWDSC is composed of 26 member agencies, which reservoir, see Izaguirre and Taylor (in press). For screening of are either cities or regional water agencies. Its two sources of samples for microcystin, the samples were shaken vigorously water are the Colorado River and water from northern and two drops of the overlying water were tested using the California, called State Project Water (SPW), delivered through ELISA tube kit. Samples that gave a strong positive response À1 the California Aqueduct. MWDSC operates three reservoirs in (43.0 mgL ) were used for the isolation of the toxin producer. Riverside County: Lake Mathews, Lake Skinner, and Diamond A small amount of well-mixed sample was added to 30 or Valley Lake (DVL). The former is the terminal reservoir of the 50 mL of a modified BG-11 medium (Stanier et al., 1971) with À1 Colorado River system. The other two reservoirs are supplied sodium nitrate at 100 mg L , and cycloheximide added at À1 with a blend of the two waters. In addition, the state 20–40 mg L , in erlenmeyer flasks. The cultures were incu- Department of Water Resources owns and operates Lake bated at 25 1C in an 18 h light/6 h dark cycle, at approximately À2 À1 Perris, a combined drinking-water and recreational lake that 6.9–7.9 mEm s . Flasks were shaded with white paper receives SPW. Lake Mathews is closed to the public, while cylinders to reduce incident light. After 2 days, small clumps Lake Skinner and DVL permit boating and fishing but no body of growth were transferred to an agar medium prepared by contact with the water. Swimming and bathing are allowed in adding the following ingredients to membrane-filtered À1 Lake Perris. (0.45 mm) raw water (SPW) in mg L : NaNO3 100, K2HPO4 40, In the summer and fall of 2001, a number of planktonic and ferric ammonium citrate 3, with purified agar (Difco, À1 cyanobacterial blooms occurred in two reservoirs, Lake Sparks, Maryland) 1%w/v and cycloheximide 20 mg L . Agar Skinner and Silverwood Lake (which consist of SPW). The plates were incubated under the same conditions as the results of analyses performed by Dr. Gregory L. Boyer, SUNY, liquid cultures, and after several days filaments that migrated Syracuse, NY indicated high microcystin concentrations in away from the inoculum were isolated using a 25 mL micro- surface bloom material from Lake Skinner (mean of two pipette and placed in a small volume of modified BG-11 samples was 304 mgLÀ1), and prompted the development of a medium as described in Izaguirre (1992). These cultures were cyanotoxin monitoring program at MWDSC in 2003. Monitor- allowed to develop and then tested for microcystin by the ing involved the use of two ELISA test kits (Envirologix Inc., ELISA tube method using the Envirologix kit mentioned Portland, ME) for the testing of water and attached algal above. samples. In the course of monitoring, it was found that benthic algal samples from Lake Mathews were positive for 2.2. Analysis of microcystin microcystin. Subsequent sampling of the three other reser- voirs, Lake Skinner, DVL, and Lake Perris, also yielded Cultures that tested positive for microcystin by the tube assay microcystin-positive samples. The initial positive samples were examined microscopically and morphological charac- from Lake Mathews contained a variety of filamentous teristics noted. These cultures served to inoculate 30 mL cyanobacteria (including Oscillatoria, Phormidium, Lyngbya, cultures for microcystin analysis. Cultures were harvested for Pseudanabaena, and Spirulina spp.), the colonial Aphanothece, microcystin analysis by measuring the volume and filtering as well as diatoms. Originally, Aphanothece was thought to be through a glass fiber (934-AH, Whatman Inc., Clifton, NJ), and the microcystin producer due to its relation to Microcystis; frozen until shipment. Analyses were performed at the however, further samples revealed no link between micro- laboratory of Dr. Gregory L. Boyer (College of Environmental cystin and this organism. All positive samples had in Science and Forestry, SUNY, Syracuse, NY), under contract common a benthic green filamentous morphotype belonging with MWDSC. Filters were extracted with 50% acidified to the order Oscillatoriales, approximately 10–15 mmin methanol using ultrasound. Microcystin content was diameter with untapered apices. verified and quantified by means of the protein phosphatase Download English Version: https://daneshyari.com/en/article/4486879 Download Persian Version: https://daneshyari.com/article/4486879 Daneshyari.com.