Towards the development of environmentally friendly methods to control harmful blooms of Prymnesium parvum



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Prymnesium parvum (Prymnesiophyceae) is a harmful algal bloom- (HAB)-forming species known as golden alga in North America. This species secretes toxins that are lethal to many aquatic organisms including fishes, amphibians, crustaceans, shellfish, and other algae. Prymnesium parvum has been identified in marine, estuarine, or brackish inland habitats globally. In the USA, it is found in 25 states and toxic blooms have been reported in 21 states. Blooms of Prymnesium parvum have had considerable ecological and economic impacts worldwide. Despite its widespread harmful effects, effective, practical methods to control golden alga blooms in the field are unavailable. Recent studies on methods of HAB control have focused primarily on the use of plant-based products due to their high biodegradability, presumed short half-life, and effectiveness as inhibitors of algal growth. However, little research has been done on the use of natural products to control P. parvum growth. Earlier studies examined the effects of barley straw (Hordeum vulgare) preparations on this species, but the results were not promising. A plant species that is the target of increasing attention as a potential source of natural algicides is Arundo donax, a graminaceous plant commonly known as giant reed. Giant reed is a highly invasive riparian plant native to eastern Asia, that was introduced to North America in the early 1800s. Various extract preparations from giant reed can inhibit growth of other HAB species such as the cyanobacterium Microcystis aeruginosa and the marine dinoflagellate Karenia brevis as well as golden alga. Several potential allelochemicals have been identified in methanolic and water extracts of giant reed. Two of the identified allelochemicals, Gramine and Skatole, also showed growth-suppressing activity against P. parvum. Gramine and skatole, however, were relatively less potent than whole extract of giant reed when their estimated relative content was accounted for, suggesting the existence of additional potent allelochemicals in the extract. All these results suggest that giant reed and giant reed derived allelochemicals could be a potential source of natural products for controlling golden alga blooms. Preparation of giant reed extract, however, is a complicated and expensive process. A simpler and less costly preparation, such as a water extract (leachate), may be more appealing to water managers. The unintended effects of any bloom control products on nontarget organisms should be characterized before they are applied in the field. There is some evidence indicating that, among microalgae, growth inhibition in the presence of Arundo extracts is observed against HAB species such as M. aeruginosa but not in some species of green algae. The molecular genetic mechanisms underlying Arundo's allelopathic activities reported by previous studies (e.g., against M. aeruginosa) are unknown. A better understanding of how natural products work to control HABs might help in the design of efficient treatments that achieve the desired result with minimal side effects. Based on the preceding literature review, the following objectives were established for this project: • Objective 1. To determine the effect of untested allelochemicals identified in extracts of A. donax on golden alga growth. Working hypothesis: there are Arundo allelochemicals that have not yet been tested in P. parvum that will have greater potency than those tested in previous studies. • Objective 2: To determine the effect of Arundo chips and leachate on golden alga growth. Working hypothesis: both preparations will be effective algicides against P. parvum. • Objective 3: To determine the toxicity (spectrum of activity) of selected preparations on non-target organisms. • Objective 4: based on the results of Objectives 2 and 3, to determine the underlying molecular mechanisms for the inhibitory growth activity of Arundo leachate on P. parvum. Working hypothesis: Arundo leachate targets the expression of P. parvum genes involved in cell growth (division). Objective 1 is addressed in chapter 2. We examined five natural compounds present in the invasive plant Arundo donax and one synthetic derivative (5,6-dichlorogramine) for their effect on P. parvum growth. All compounds except one inhibited growth in the following order of potency: ellipticine>>5,6-dichlorogramine>1 H-indole = 2,4,6-trimethyl-benzonitrile>gramine. Ellipticine was by far the most potent inhibitor, with full algicidal activity at concentrations as low as 0.04 mg L-1 and 3- and 9-day IC50 values of 0.012 and 0.007 mg L-1, respectively. A reduction in chlorophyll content and swimming activity and an increase in length and volume (swelling) were documented in algal cells exposed to 0.01–0.02 mg ellipticine L-1. These results show that ellipticine is among the most potent natural algicides identified to date. The sixth compound tested, oleamide, unexpectedly stimulated algal growth above control levels. Overall, these observations confirm the existence of highly potent anti-P. parvum allelochemicals in giant reed and demonstrate potential for using products derived from this plant in the development of natural, environmentally friendly methods to control harmful algal blooms. Objectives 2 and 3 are addressed in chapter 3. We first examined the effect of Arundo dried chips and leachate on P. parvum growth. Giant reed chips immersed in culture medium at the start of the cultures showed complete algicidal effect at ≥ 8 g l-1, and its IC50 value at 24 h was 3.1 g l-1 (± 0.8). Leachate prepared in advance of the experimental cultures also had an algicidal effect at 3 g l-1. We then determined effects of leachate and ellipticine on growth of P. parvum and the green microalga Chlorella sorokiniana; survival and reproduction of the planktonic crustacean Daphnia pulex; and hatching success and larval survival and behavior of the teleost fish Danio rerio. Leachate from 3 g chips l-1 stimulated growth of C. sorokiniana but was lethally toxic to D. pullex and P. parvum, and mildly affected D. rerio behavior. We lowered the concentration of to 1 g l-1 and at this concentration leachate had no effects on D. rerio, moderate effects on D. pulex reproductive output, and fully suppressed P. parvum growth. Ellipticine at 0.01 mg l-1 had no effects on D. rerio, slightly delayed reproduction in D. pullex, acutely but reversibly inhibited C. sorokiniana growth, and irreversibly inhibited P. parvum growth. These observations suggest that when applied at appropriate concentrations, natural products derived from Arundo donax can effectively inhibit P. parvum growth with little or no harm to nontarget organisms. Objective 4 was addressed in chapter 4. Prymnesium parvum were exposed to Arundo leachate for 24 hours and the effects on growth were determined by measuring cell density and those on transcriptome composition were evaluated by differential gene expression. Differentially expressed transcripts were analyzed with Gene Ontology and KEGG pathway methods to determine cellular processes and pathways enriched with these transcripts. The results showed that leachate exposure completely blocked cell division (growth) and strongly downregulated processes and pathways associated with cell division, membrane transport, and photosynthesis. The relevance of the disruption in cell division and photosynthesis (necessary for carbon fixation) functions to the reduced growth of P. parvum is self-evident. Most pathways within the membrane transport category were related to ATP-binding cassette (ABC) transporters, which are necessary to maintain homeostasis during biotic and abiotic stress—thus, downregulation of ABC transporters may also have contributed to impaired growth. The information obtained by this study has improved our understanding of how Arundo leachate suppresses growth of P. parvum and may facilitate the development of bloom control methods based on this natural product.

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Harmful algal blooms (HABs), 5,6-dichlorogramine (DCG), Dimethyl sulfoxide (DMSO), Kyoto Encylopedia of Genes and Genomics (KEGG)