“Zebra mussel and Daphnia regulate the Microcystis-rich phytoplankton appearance in Lake Ontario by way of selective feeding rejection”
Introduction and problem description
According to Christoffersen’s classical theory phytoplankton-zooplankton interaction is one of the contributory factors determining the primary and secondary productivity and fauna diversity in the littoral zone of aquatic ecosystems, including the oligotrophic lakes of the Arctic region (42-50). Zooplankton that varies from minute flagellated protozoan to large insect and fish larvae, feed on bacterioplankton and phytoplankton (mostly <30µm size) comprising green microalgae and cyanobacteria. In freshwater lakes, meso- (0.5-1mm) and megazooplankton (>1mm) belonging to the taxa copepods, decapods and mollusks are the major constituents of zooplankton populations. However, as and when cyanobacteria dominate over other plankton species due to inflow of domestic and agricultural run-offs rich in phosphorus from the catchments and consequently organic matter increases leading to eutrophication, in most occasions as observed by Gilbert, smaller cladocerans and rotifers dominate as prominent zooplankton (1727-40). Further studies reveal that larger cladocerans, Daphnia etc, ingest more cyanobacteria than other phytoplankton and are negatively affected, consequently affecting the entire zooplankton community structure, secondary production and as a whole the flow of energy at higher trophic level. Lack of grazing built up heavy cyanobacterial population to a proportion nascent/toxic to aquatic animals and humans, called “water blooms” (Codd et al 1-138). Many factors determine the selective grazing suppression of cyanobacteria like the morphological and mucoidal colonial features interfering with ingestion, lack of essential fatty acids like PUFA, sulfide-like odor, and number of grazing-deterrent substances include toxins (Thostrup & Christoffersen 447-467). It has been a pertaining issue that microcystins, a group of over 70 cyclic peptide toxins produced from a prominent cyanobacterial component of “water blooms” world over, are the most dreaded aquatic toxin for human and animal life. About 40% of Microcystis spp. in water blooms produce these toxins threatening the quality of water. Fishes and many aquatic mammals are subject to exposure to this toxin primarily affecting liver. It has been a debatable issue whether microcystins are also responsible for aquatic crustacean copepods’ and decapods’ deaths. It was more recently discovered that many other peptides inhibiting the digestive proteases in Daphnia gut are crucial in reducing appetite for cyanobacteria. Since green microalgae are not potential repellants, they are preferred so far as grazing is concerned.
Mollusks such as zebra mussels (Dreissena polymorpha) also feed on different species of phytoplankton communities. As surveyed by Brittain and co-workers in oligotrophic brackish water Lake Ontario this is abundantly present (241-249). The overall quality is considered to be poor due to high sediment and nutrient especially P loading from surrounding watersheds spread in large areas leading to eutrophic conditions, making the lake unfit for recreational activities. Increase in Microcystis-dominant cyanobacterial populations, siltation and lack of oxygen for deeper fauna has started to further deter the quality of the lake in coastal regions. According to Bykova and co-workers a course of measures did prevent the bloom formation, but since last decade there has been re-emergence of cyanobacterial phytoplankton community particularly of Microcystis spp. and Increase in number of zebra mussels has been attributed to the unprecedented rise in this toxic cyanobacterium (362-72). Unlike the crustacean-cyanobacterial interaction the mollusk-cyanobacterial interaction is quite unpredictable and complex. According to Vanderploeg and co-workers zebra mussel’s selective filtration promotes toxic cyanobacteria (Microcystis) blooms (1208-21). Another report by Sarnelle et al suggests that zebra mussels exert both adverse and stimulatory effects on population growth of toxic Microcystis, and this relates to the availability of phosphorus in the water body. In this interesting piece of work it was shown that phosphorus dynamics regulate the detrimental effect of Microcystis on mussels. When P content was low relative to N the mussels started to graze toxic Microcystis. Conversely, higher phosphorus content suppressed mussel growth from toxic cyanobacteria owing to feeding inhibition (896-904). In another report from Bykova and co-workers, feeding of debris rich in N, P and organic matter by zebra mussels enable clearing/consumption of N and carbon but release of phosphorus. This accumulated phosphorus and increase in P/N ratio in water in fact promote Microcystis growth (362-72). Moreover, selective filtration and rejection of live cyanobacterial cells in pseudofeces of zebra mussels also promote Microcystis bloom formation (Juhel et al 810-16). There are contradicting records available from the work of Pires and co-workers that, both microalgae and cyanobacteria (Microcystis spp.) are equally preferred diets for zebra mussels and there is practically no discrimination (116-26). It is still not clear whether microcystins and other peptides which have deterrent effects on Daphnia also suppress filtration rates in zebra mussels. Therefore, it was a matter of interest to establish and ascertain the cause of deterrent inhibition (if any) of toxic cyanobacteria e.g. Microcystis spp. on both Daphnia and Dreissena.
Objectives and hypotheses
The primary objective of the proposal is to clearly elucidate the role of mussels in either eliminating or promoting the abundance of Microcystis spp. or having no impact. Moreover, addition of Dreissena in water body would affect anyway the already prevailing green algal phytoplankton population such as the dynamic changes in abundance or biovolume, by way of selective rejection of grazing of the cyanobacterial planktonic cells rich in Microcystis spp. if an alternate green microalga is already available as food? If infield grazing inhibition prevails due to cyanobacterial ingestion in mussels then in lab this effect will be correlated in two different herbivorus zooplankta, Dreissena and Daphnia. In addition, putative grazing deterrent substance(s) would be partially extracted and tested on Daphnia life table to extrapolate any toxic effect on zebra mussels.
We hypothesize that (a) Microcystis spp. exerts a direct grazing suppression and toxic effects on population growth of both the crustacean and mollusk zooplankta identically. We also hypothesize that (b) toxic substances are produced and accumulated within the cyanobacterial cells that repel zooplankton, and these attributes profoundly affect the biovolume and abundance of green algal species in the phytoplankton community in oligotrophic lakes. One of the additional underlying hypotheses would be (c) whether indeed P and N content has some effective detrimental/beneficial role to play towards mussel growth, and can this attribute be used for effective application of mussel rearing, aiming to remove toxic Microcystis blooms.
Consequently, we would try to establish if any correlation exists between increased number of zebra mussels and re-emergence of Microcystis spp. in Lake Ontario. If our hypothesis is validated then an overall impact would be that both zooplankta would have negative association with cyanobacteria and either zooplankton would perish or prefer micoalgae as alternate food source. If productivity is imbalanced, toxic cyanobacteria, would take over the green phytoplankton and overall quality of the lake would deter further. Toxic substances like microcystins, the cyclic peptides, produced from such blooms and foul odor would badly affect the habitat and health of aquatic fauna, fishes, domestic animals and human in the catchments. Recommendations for possible remedial measures will be given for future courses of action.
Study Area/Methods and Experimental Design
Sampling Sites: At least 7 stations in Lake Ontario Northern bay at water column depth of 10-15m would be identified considering some basic information on phytoplankton abundance and selecting sites with paint-like thick surface scums of Microcystis spp. Some prior information of clarity, dissolved O2, BOD and chlorophyll density etc. also would be desirous.
Collection of zebra mussels: Zebra mussels will be separated from gravel, wooden logs etc. and allowed to stick to plastic substrates suspended in lake water. Average mussel length and weight will be recorded and about 10-15 animals will be suspended in each enclosure tube above the mesh.
Fabrication of tubular microcosms: Basically the fabrication strategy of Bykova and co-workers (365) would be applied. Tubular polyethylene enclosures (2.5m diameter, 10m depth) for experimentation with mussels, open at top and immersed in soil/sediment level from bottom, will be installed in June-July every year and will continue to be used year-round until water freezes. Large volume of lake water will be filtered through 200µm mesh to separate zooplankton, insects etc. and such water of different compositions will be pumped once every 10-15 days from bottom to top until the tubes are filled. Inside the tubes the water will pass through 1mm mesh filters attached towards bottom on which mussels will be placed. This mesh will be placed such that water can move only upwards, and beneath there will be a valve to stop downward flow. This fabrication would incur special installation and recurring expenses. After three cycles of water replenishment the mussels will be shifted to lab for feeding experiments.
Collection and storage of Microcystis spp. phytoplankton: Surface water film/scum will be filled in large plastic bottles and buoyant colonies will be allowed to float, which can be removed using Pasteur pipettes and lyophilized to dryness until used. For experiments large quantity of material in dried form will be collected from different sites of the same lake, pooled and preserved. As required they will be suspended in filtered lake water and hand homogenized to disrupt the colonies. The cells will be used for feeding and other experiments.
Phytoplankton abundance and biovolume determination: Epilimnetic water samples of 1m deep, to be pumped in tubular microcosms, will be fixed in Lugol’s solution and examined by high power inverted microscope for identification up to genus using taxonomic keys. Abundance (number of cells/colonies/filaments) and biovolume (cell volume) for each identified genus would be enumerated and for this Neubauer hemocytometer can be used. From the biovolume biomass can be calculated from standard conversion.
Treatments: In the microcosm experiments the abundance and/or biovolume of phytoplankton in different sets of experiments will be monitored prior to and after every round of change of water. The filtered (200µm) lake water will be mixed with graded dried and homogenized biomass of Microcystis spp. or kept without any mix (controls). Additional treatments for mussels would be phosphate and ammonia supplementation to above water by adding defined weights of fertilizers. For every batch of treatment separate tubular microcosm will be established and treatment would continue for 30 d. At the end of treatment phytoplankton abundance, biovolume and biomass will be determined. This will ensure if there is any kind of preferential grazing of algal or Microcystis phytoplankton. The P and N addition would determine how water chemistry influence preferential grazing.
Manpower requirement: For maintaining the microcosm one part-time helper and for collection of material and preparation of water samples one trained technical assistant would help the doctoral student.
Algal cultivation: Green algae Scenedesmus and Ankistrodesmus are routinely used as feed for Daphnia and mussel. These cultures will be procured from culture collection centers and grown in lab inappropriate mineral media. Cells will be harvested by filtration/centrifugation and enumerated using hemocytometer.
Establishing Daphnia population: Daphnia eggs can be procured from collection center and these can be hatched in diluted EDTA and transferred to medium containing Scenedesmus cells at appropriate density suspended in 30µm mesh filtered and aerated lake water (majority of phytoplankton removed). The population can be raised in beakers at 15oC. Water can be changed every two days and population increase is enumerated by counting the individuals.
Mussel feeding experiment: In the field treatment experiments some mussels would be dead. The live individuals will be used for feeding experiments. To ensure that enclosed mussels are healthy and actively feeding mussels along with smaller substrates will be hanged in beakers filled with suspension of Ankistrodesmus in 30µm filtered lake water and allowed to feed for 4-5 h. Feeding rates can be calculated by comparing initial and final concentrations of algal chlorophyll. Valve and siphon movement is another criterion to ascertain filtration ability. Mortality will be established within 7 d of feeding experiment and its percentage will be recorded. Daphnia feeding experiment: Along with routine algal feed different combinations of dried material of Microcystis spp. will be supplemented in population growth studies. Phytoplankton concentrate (filtered by GF/C filters) will also be mixed with routine algal feed. The difference in chlorophyll density in 6-8 h will be measured to determine the filtration rates. One of the sophisticated methods to make differential counts of green vs. cyanobacterial cells is flow cytometry and this procedure can be adapted here (Pires et al 119).
Population growth experiment: Food types for Daphnia individuals will be green alga alone, green alga and filtered phytoplankton and green alga and Microcystis scum at different proportions. At every 2 days live animals will be counted and transferred to identical food types and this process will continue for 14 days to get life table for each food type.
Toxicity tests: This test can be performed only with Daphnia. For this a crude extract of Microcystis spp. will be prepared by sonication and centrifugation, and aliquots supplemented to starved daphnids. Individuals fed on normal algal diet will be transferred to autoclaved water without any food. The concentration of extract that causes mortality higher than starvation will be considered toxic. The extract concentration-dependent % mortality would be plotted and from PROBIT analysis the 50% lethal concentration can be enumerated. The same tests can be performed in aquaria filled with autoclaved water, and certain number of healthy mussels will be hanged using plastic substrates. Different concentrations of Microcystis spp. extract and control without extract will be added and % mortality will be evaluated in each set.
Manpower requirement: One doctoral student scholar and one technical assistant would carry out the lab experiments.
|2010||1st||Site selection, mussel and Microcystisspp. collection, lake water chemical analysis, microcosm fabrication, installation, method standardization, Manpower recruitment, material purchase|
|2nd||Microcosm operation, mussel rearing in tubes, Algae culturing, freeze-drying of Microcystisspp. Continuation with above activities|
|3rd||Mussel treatment experiments, phytoplankton enumeration, biovolume, other microcosm assays|
|4th||Continuation with 3rdq. activity, data collection of treatment tests|
|2011||1st||In situmortality assays, mussel collection for feeding trials, data collection and analysis of treatment tests|
|2nd||Daphniaprocurement and establishment of population, data analysis, continue collection and freeze-drying of Microcystisspp.|
|3rd||Daphniafeeding experiments, Daphnialife table experiments, data collection and analysis|
|4th||Second round of mussel treatment experiments and feeding trials, phytoplankton analysis|
|2012||1st||Continuation with previous q. activity, Microcystisspp. extract preparation|
|2nd||Daphnialife table experiment, toxicity tests, mussel toxicity tests, data collection and analysis|
|3rd||Repeat toxicity tests, assembling all processed data, preparation of research report for agencies, research publication|
|4th||Workshop and seminars, group meetings, publication, report preparation|
A successful completion of the aquatic ecology project would ensure new directions in research aiming at environmental biotic interactions like allelopathy between zooplankton and phytoplankton species. In particular the following output would be beneficial:
- New finding – the confusion on the implications of zebra mussels in regulating the proliferation of toxic cyanobacteria in Great Lakes would be partially resolved. A role of chemical composition of water in above relationship would be deciphered. The precise anti-grazing component of Microcystis common to co-inhabiting crustacean and mollusk zooplankton would be established. The fate of selective grazing on overall phytoplankton dynamics at different P and N levels would be known.
- Value of project – Mussels is delicious food item. If toxic cyanobacteria are ingested the toxins would enter the food chain and mussels would not be exception to accumulate toxins. In the higher trophic level aquatic life forms feeding on mussels would be affected. The project aims to determine these factors of concern and tries to manipulate the chemical conditions or phytoplankton abundance such that toxic cyanobacteria would be grazed with no disturbance to the aquatic fauna.
- Contribution for solving problems – Microcystis is a potentially threatening toxic cyanobacteria occurring globally. All forms are not toxic and chemical assays for toxin detection are expensive and not always foolproof. In this project bioassays will be established in which feeding inhibition using two zooplankta and life table experiments and toxicity assay using Daphnia would help detect emergence of toxic forms in recreational waters.
Another application of the project outcome would be to effectively maneuver the nitrogen and phosphorus content such that zebra mussel can be reared to graze on toxic Microcystis spp. and this will be an effective method to eradicate this problem. Care should be taken not to market these animals for human consumption as they may still contain microcystins.
|a) Ph.D. student researcher (1; mm* = 36)||24,000||24,000||24,000|
|b) Field/Lab assistants (2; mm = 30)||15,000||15,000||15,000|
|e) Materials (Equipment** + Consumables)||6,000||1,500||1,500|
Man months: Equipment (Planktonic nets, Lyophilizer, Inverted microscope, Flow cytometer, BOD incubator etc) + fabrication of tubular enclosures, other minor devices.
- Amount from Ryerson Univ. $50,000
- Amount from WWF $50,000
- Amount requested from other agencies $105,200
Brittain, Scott M., et al. “Isolation and Characterization of Microcystins, Cyclic Heptapeptide Hepatotoxins from a Lake Erie Strain of Microcystis aeruginosa.” Journal of Great Lake Research 26.3 (2000): 241-249.
Bykova, Olga, et al. “Do zebra mussels (Dreissena polymorpha) alter lake water chemistry in a way that favours Microcystis growth?” Science of the Total Environment 371 (2006): 362–372.
Christoffersen, Kirsten. “Ecological implications of cyanobacterial toxins in aquatic food webs.” Phycologia 35 (1996): 42-50.
Codd, Geoffery A., et al. “Cyanonet, a global network for cyanobacterial bloom and toxin risk management: Initial situation assessment and recommendations.” UNESCO-IHP-VI Technical Documents Hydrology Series 76 (2005): 1-138.
Gilbert, J.J. “Differential effects of Anabaena affinis on cladocerans and rotifers.” Ecology 71 (1990): 1727-40.
Juhel, Guillaume, “Introduction of Pseudodiarrhoea in zebra mussels Dreissena polymorpha (Pallas) exposed to Microcystins.” The Journal of Experimental Biology 209 (2006): 810-816.
Pires, L. M., et al. “Selective grazing by adults and larvae of the zebra mussel (Dreissena polymorpha): application of flow cytometry to natural seston.” Freshwater Biology 49 (2004): 116–126.
Sarnelle, Orlando, et al. “Complex interactions between the zebra mussel, Dreissena polymorpha, and the harmful phytoplankter, Microcystis aeruginosa.” Limnology & Oceanography 50.3 (2005): 896–904.
Thostrup, Lykke, and Kristen Christoffersen. “Accumulation of microcystin in Daphnia magna feeding on toxic Microcystis.” Archives of Hydrobiology 145.4 (1999): 447-467.
Vanderploeg, Henry A., et al. “Zebra mussel (Dreissena polymorpha) selective filtration promoted toxic Microcystis blooms in Saginaw Bay (Lake Huron) and Lake Erie.” Canadian Journal of Fisheries & Aquatic Science 58 (2001): 1208–1221.