Mimosine, the Allelochemical from the Leguminous Tree Leucaena leucocephala, Selectively Enhances Cell Proliferation in Dinoflagellates

Abstract

Mimosine, the allelochemical from the leguminous tree Leucaena leucocephala, is toxic to most terrestrial animals and plants. We report here that while mimosine inhibits major phytoplankton groups, it enhances cell proliferation in dinoflagellates. On addition to coastal seawater samples, mimosine is able to confer a growth advantage to dinoflagellates. The use of mimosine will promote the isolation and culture of this group of phytoplankton.

Leucaena leucocephala is a tropical and subtropical legume widely used in agroforestry systems throughout the world. It has been hailed as the perfect tree because it can serve many purposes, as foliage for livestock, as fuel wood, or as green manure (1). However, introduction of Leucaena outside its indigenous range has often led to acute and chronic toxicosis in animals (14). The agents of toxicity are the allelochemicalsmimosine (α-amino-3-hydroxy-4-oxo-1-pyridine propanoicacid), a nonprotein amino acid, and its main degradative product, 3,4-dihydroxypyridine (2). The concentrations of mimosine in air-dried Leucaena leaves were found to be in the range of 2.5 to 5.75% (2), and mimosine can be easily removed by soaking the leaves in water for 24 h (2). Soil extracts from around Leucaena trees are also toxic to other plants (12). We are interested in investigating whether mimosine may have allelochemical effects on aquatic phytoplankton species.

We tested the effects of mimosine at low micromolar concentrations on pure cultures of four different groups of phytoplankton: the Cryptophycea Rhodomonas salina (CCMP1319), the Prymnesiophycea Isochrysis galbana (CCMP1323), the Bacillariophycea Cylindricus fusiformis (PCC100), and the dinoflagellate Heterocapsa triquetra (CCMP449). Cultures of phytoplankters were obtained from Bigelow Laboratory for Ocean Sciences or the Plymouth Culture Collection. The cultures were maintained in f/2 medium at 18°C under a photon flux from fluorescent tubes (Phillips daylight) of 50 μmol · m⁻² · s⁻¹, and a cycle of 14 h of light and 10 h of darkness. For cell proliferation assays, exponentially growing cells were diluted 10 times with fresh medium before the addition of mimosine. The heterotrophic dinoflagellate Crypthecodinium cohnii (strain 1649 from the Culture Collection of Algae at the University of Texas in Austin) was cultured with its own synthetic medium (16) with the stated modifications during the experiment. All chemicals were from Sigma Corporation unless otherwise stated. All growth experiments were performed in triplicate. Cells in all samples were counted with a Coulter counter at least three times. With 0.01 and 0.1 mM mimosine, the numbers of cells observed for R. salina, I. galbana, and the diatom C. fusiformis (Fig. 1a to c) were lower, though not significantly lower, than the numbers observed for control cultures over the course of the experiment (8 days). At 1 mM mimosine, no increase in the number of cells was observed for I. galbana. For C. fusiformis and R. salina, the mean numbers of cells in 1 mM mimosine were significantly lower (25 and 75%, respectively) than those in the controls. For the dinoflagellate H. triquetra (CCMP449), not only did mimosine (1 mM) fail to have negative effects on cell proliferation, it surprisingly increased the cell number significantly compared to that of the control culture (Fig. 1d).

FIG. 1.

Effects of mimosine on cell proliferation of monocultures of major phytoplankton groups. (a) H. triquetra (Dinophyceae); (b) C. fusiformis (Bacillariophyceae); (c) I. galbana (Prymnesiophyceae); (d) R. salina (Crytophyceae). , control; ×, 0.01 mM mimosine; ▴, 0.1 mM mimosine; ▪, 1.0 mM mimosine.

The differential effects of mimosine on monocultures of phytoplankton groups may be translated into selective effects in mixed populations. We further tested whether mimosine can confer an advantage in the phytoplankton community by adding mimosine directly to natural seawater samples. The samples were collected from Port Shelter in eastern Hong Kong and filtered through a 100-μm mesh immediately before use to remove all zooplankton. Mimosine was then added to 2 mM, and samples were taken for estimation of the percentages of dinoflagellates and diatoms. Within 6 days, dinoflagellates became the dominant group. The group's population increased from 30 to 60% of the total population (Fig. 2). The diatoms, which were the dominant group in the control, decreased to 10% of the total population in 6 days (at the time of measurement) upon treatment with mimosine. The effects of mimosine lasted for approximately 10 days, and other phytoplankton groups recovered (data not shown) if fresh mimosine was not added at this point. To our knowledge, this is the first report of a competitive advantage conferred by a major terrestrial allelochemical to a major phytoplankton group. There are various demonstrated mechanisms of mimosine toxicity (6-8). Mimosine can affect DNA metabolism in eukaryotes (3, 15, 17) and is widely used as an agent to synchronize mammalian cells in S phase. The mimosine resistance of dinoflagellates is probably attributable to the peculiar chromatin structure and DNA metabolism of the group (9, 10). This resistance to mimosine is also the first demonstrated by a eukaryotic group. With regard to nodulation, mimosine provides a competitive advantage to mimosine-degrading Rhizobium strains (13). Inoculation of these bacterial strains into ruminants confers resistance to Leucaena toxicity (5). Mimosine is a nonprotein amino acid, and its ability to stimulate cell proliferation in dinoflagellates may be accounted for by the heterotrophy of many photosynthetic species (4). We were able to grow the heterotrophic dinoflagellate C. cohnii in a synthetic medium (16) with all its nitrogen sources replaced by mimosine, suggesting that dinoflagellates may utilize mimosine as a nitrogen source (Fig. 3). The effects of mimosine on natural seawater samples probably resulted from both the selective growth of dinoflagellates and the selective death of other phytoplanktons, as there was an actual increase in the number of dinoflagellate cells during mimosine treatment.

FIG. 2.

Effects of mimosine on mixed phytoplankton in natural seawater samples. Percentages of dinoflagellates (a) and diatoms (b) relative to the total number of phytoplankton are shown. Seawater samples were filtered through a 100-μm mesh before the experiment. , control; ▪, 0.2 mM mimosine; ▴, 2.0 mM mimosine.

FIG. 3.

Cell proliferation of the heterotrophic dinoflagellate C. cohnii in medium with nitrogen sources replaced by mimosine. A 1/10 dilution of exponentially growing C. cohnii cells was inoculated into MLH¹⁶ medium as the control ( ); into MLH without the nitrogen sources botaine, histidine, and ammonium salt (▪); and into MLH without the nitrogen sources but with either 0.01 (×) or 0.1 (▴) mM mimosine.

Biological, physical, and chemical factors as well as their interactions culminate in the development of algal blooms (11). Allelopathic interactions have long been suggested to play a role in the community structure and succession of phytoplankton groups (12). In the present study, the sensitivities of the phytoplankton groups to mimosine (1 mM) are in the order of Prymnesiophyceae > Cryptophyceae > Phaeophyceae > Dinophyceae. More species have to be tested to identify any possible trend. The present study also suggests that DNA replication can be a potential target for allelopathic interactions in phytoplankton groups. The dinoflagellates are major causative agents of red tides or harmful algal blooms. Occurrences of red tides after precipitation have been reported to occur in many coastal areas (19). Given the high concentration of mimosine in Leucaena and its high production rate, it is conceivable that in coastal areas with Leucaena plantation, mimosine may be one of the factors contributing to the formation of dinoflagellate blooms. Further oceanographic investigations are required to delineate possible relationships between mimosine concentrations and dinoflagellate blooms. The difficulty of culturing many dinoflagellate species under laboratory conditions is a major factor that hinders research into this group of phytoplankton. The use of mimosine can greatly enhance the growth of many dinoflagellate species in mixed populations. Using this selective property of mimosine, we were able to isolate pure dinoflagellate strains from different genera, including Gymnodinium, Karenia, Prorocentrum, and Protoperidinium, from natural seawater (unpublished data). While there is a biotechnological potential in the degradation of toxic mimosine by heterotrophic dinoflagellates, the use of Leucaena meal in aquaculture feeds (18) has to be further considered with caution in coastal areas with harmful algal blooms.