Molecular Identification Of Sessile Ciliated Protozoans Biology Essay

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Fens, as unique wetland systems, have been destroyed mostly for agricultural reasons, leaving only 2 percent remaining. There are currently 200 fens located in Iowa. Ostracods are a regular inhabitant but are usually overlooked in aquatic environments such as fens. Ostracods can serve as indicators of change in wetland systems because they have a high response threshold to disturbance. The proposed research will be focused on Ciliates in the family Lagenophryidae living on three species of ostracoda (Candona candida, Cypridopsis aculeata, and Eucypris rava), which were collected from two fens in Buena Vista County, Iowa.  Parasite effects on host organisms range from minor harm, allowing the host to live and reproduce normally and complete its normal life cycle, to completely interfering with reproduction or causing premature death of the host. The goal of this study is to describe biodiversity and abundance of freshwater sessile ciliated protozoans infesting ostracods in fens. The second goal of this study is to utilize Qdots to investigate nutrients such as nitrogen, phosphorus, and potassium influence on the abundance or absence of ciliated protozoans on the carapace of freshwater ostracods. The last goal of this study is to determine the overall impact the presence that the ciliated protozoan's are having on ostracod overall health and fecundity utilizing metabolomics.

Introduction (20 points)

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The ciliated protozoan is in the phylum ciliophora and the kingdom Protista (Lynn 2007). It can be defined as sessile or motile unicellular heterotrophy (Lynn 2007). This group comprises a largely diverse group of organisms with over 5,000 described species (Lynn 2007). An Ostracod is in the subclass ostracoda and in the class Crustacea. This organism is a small bivalve unsegmented organism (Lynn 2007).

Both ostracods and ciliated protozoans are thought to occur in virtually all water environments. Their cosmopolitan distribution suggests that they are an integral part of the ecosystem in which they live (Hartmut 1972).

Fens are peat accumulating wetlands receiving some higher alkalinity groundwater from a mineral substrate (Kalff 2002). As a result, fen water has a higher nutrient content, and supports macrophyte vegetation which is usually dominated by sedges (Kalff 2002). Fens contain ample amounts of biodiversity (Kalff 2002). They harbor over 200 plant species, 20 of which are considered endangered or threatened in Iowa (Bedford & Godwin 2009). Like most wetlands, only few native fens remain in Iowa (Bedford & Godwin 2009). The vast majority of Iowa fen degradation is a result of agricultural practices. Throughout the Midwest, fens have been lost through drain-tiling (Bedford & Godwin 2009).

It has been recently found that ostracods are excellent organisms to use as indicators of water quality. Ciliated protozoa have been identified as a major microbial transfer of phosphorus to higher tropic levels (Hartmut 1972). The impact generated from anthropogenic affects to our natural habitats affects ostracod richness and ciliated protozoan abundance (Hartmut 1972). Anthropogenic affects alter the habitat for ostracods and protozoan's by causing changes in the chemical, biological, and physical structure altering aquatic ecosystems functioning ability (Hartmut 1972).

The degradation of fens has had and will continue to have a long lived negative impact on fen health (Kalff 2002). These systems harbor some of the most threatened organisms yet little is known as to how to asses this degradation. Previous research conducted focused on the presence of ciliated protozoans on ostracods and the effect the ciliates were having on ostracod health. This research was conducted over the course of three years. Ostracods moved from being 13% infested to 100% infested over the course of only one year. Currently the death rates of ostracods are reaching nearly 70%.

Identification of sessile ciliated protozoans has been achieved in the past through morphological features utilizing a microscope (Lynn 2007). It is difficult to rely on morphological techniques due to the issue of phenotypic plasticity (Dopheide 2008). Molecular techniques utilizing PCR specific primers will offer more accurate and reliable for identification of sessile protozoan communities (Dopheide 2008).

The main objective of this research therefore will be to utilize PCR specific primers from the literature to find the diversity of sessile ciliated protozoan's infesting the ostracods in fens. RFLP will be utilized to create a species accumulation curve. Once this is completed the next objective of this study will be to identify the reason why the over infestation of these ciliated protozoan's are occurring on ostracods in fens utilizing Q-dots (Michalet 2005) . The last objective to study is the overall effect that the ciliated protozoan is having on the ostracods. This will be completed utilizing metabolomics (lear 2008).

General and specific goals (10 points):

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Describe biodiversity and abundance of freshwater sessile ciliated protozoans infesting ostracods in fens. The processing of this data will allow me to create a species accumulation curve.

To investigate nutrients such as nitrogen, phosphorus, and potassium influence on the abundance or absence of ciliated protozoan's on the carapace of freshwater ostracods.

Determine the overall impact the presence that the ciliated protozoan's are having on ostracod overall health and fecundity.

Methodology (30 points):

Ostracod Sample Collection:

Everything used during this study will be autoclaved prior to use to avoid contamination. The sampled ostracods will be placed in sterile water. Utilizing a pipette the ostracods will be moved though seven different water baths to attempt to move as much as the debris as possible. Micromanipulation of the sessile ciliated protozoans will be removed using a thin needle and then placed in an epindorfe tube. The Epindorfe tube will then be placed into the freezer to save for DNA extraction.

PCR amplification and purification

Primers 384F/1147R and 121F/1147R are known sequences that have been used on stream protozoans and will be used in this study as well (Dopheide 2008).PCR amplification will be done using the standard 50-micro l reaction. PCR will be utilized to detect the presence or absence of sequences using a 1% agarose gel stained with ethidium bromide (Dopheide 2008).

Cloning, RFLP analysis, and sequencing

PCR products obtained from sessile ciliates DNA using Ciliate specific primers were cloned using a TOPO TA cloning kit according the instructions (Dopheide 2008). The clones using the ciliate specific primer sets will be incubate and use the PCR standard procedure (Dopheide 2008). The cloned DNA will then be used utilizing RFLP to create a species accumulation curve by applying the different number of clones into a table (Dopheide 2008). The different species identified using RFLP analysis will be sequenced and then placed into BLAST to see if there will be any match's in GenBank (Dopheide 2008).

Q dots

Q dots will be tagged to specific nutrients such as nitrogen, phosphorus, and potassium (Michalet 2005). This will allow us to see nutrient uptake by the ciliated protozoans (Michalet 2005). The use of this technique will aid us in the understanding of how nutrients within freshwater systems impact the abundance and infestation of freshwater ciliated protozoans on the carapace of ostracods (Michalet 2005).

Metabolomics

The use of metabolomics will be applied to understand the physiological challenges the ciliated protozoans will have on the ostracods (Camoacho 2005). Metabolites will be measured on ostracods with parasite present and on ones that do not. The comparison should yield differences in metabolites produced giving an indication of ostracod stress (Camoacho 2005).

Expected results (15 points):

It is to be expected that we will find a large amount of diversity in ciliates on our ostracods. From preliminary research we believe that the family Lagenophryidae and Epistylididae will be the most common sessile ciliate to be found (Clamp 1973). We also expect that metabolomics will indicate that the infestation of the ostracods is having a long term negative impact on the fecundity of the ostracods (Clamp 1973; Herman 1971). Figure one is showing the very obvious impact that these ciliates are having on the ostracods.

We believe that the Q-dot analysis will yield the results that nitrates within the system are the largest contributor to the infestation of these ciliated protozoans on ostracods (Herman 1971). We believe this to be answer since the fen's that have the presence of the ciliated protozoan's are highly degraded areas with a large nutrient input.

Potential limitations (10 points):

As with any molecular research there will be troubleshooting. This research to the best of my knowledge has yet to be conducted on ostracods in freshwater fens. It will take some time to adapt the protocol to work for this experiment. The other limitation for this research is the difficulty that will come with removing the sessile ciliated protozoans from the ostracod shells. This will produce some contamination of ostracod DNA.

It has been known for quite some time that Q dots are in limited use in aquatic environments. This is due the hydrophobic nature of the Q dots outer shell. Ciliated protozoan's live in aquatic environments and cannot live without the presence of water. It will be difficult to find and utilize a Q dot that is versatile and non toxic in nature for the application to be successful (Michalet et al., 2005)

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Ostracods and the ciliated protozoans infesting the outside of their carapace are fairly undocumented and understudied organisms. Limitations with the use of metabolomics could be limited due to the lack of knowledge known about the metabolites ostracods produce. It will be difficult to determine which metabolites change in response to the presence of the ciliated protozoan. Standardized protocols will have to be created first to understand the change in metabolites produced during the infestation.

Reference (5 points):

Bedord BL, Godwin KS. (2009) Fens of the United States: Distribution, characteristics, and scientific connection versus legal isolation, Wetlands. 23, 608-629.

Camoacho D, Fuente A, Mendes P. (2005) The origin of correlations in metabolomics data, Metabolomics. 1, 53-63.

Clamp J. (1973) Observations on the Host-Symbiont Relationships of Lagenophrys lunatus Imamura, Department of Zoology. 20(5) 558-561.

Dopheide A, Lear G, Stott R, Lewis G (2008) Molecular Characterization of Ciliate Diversity in Stream Biofilms. Applied and Environmental Microbiology, 74, 1740-1747.

Hartmut B. (1972) Ciliated Protozoa, An illustrated guide to the species used as biological indicators in freshwater biology, 1st edition, 1-198.

Herman S, Coull B, Brickman L. (1971) Infestation of Harpacticoid Copepods (Crustacea) with ciliate Protozoans, Journal of Invertebrate Pathology. 17, 141-142.

Lear G., Anderson MJ., Smith JP., Boxen K., Lewis GD. (2008) Spatial and temporal heterogeneity of the bacterial communities in stream epilithic biofilms. FEMS Microbiology Ecology. 65(3) 463-473

Lynn DH (2007) The Ciliated Protozoa. Characterization, Classification, and Guide to the literature, 3rd edition, 1-605.

Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li J, Sundaresan G, Wu AM, Gambhir SS, Weiss S. (2005) Quantum dots for live cells, in vivo imaging and diagnostics. Science 307:538-544.