Revealing Diversification Of Zooplankton Biology Essay

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Our study focuses on documenting the zooplankton community structure of two South Australian reservoirs, Myponga and South Para, by means of genetic and morphological data. Sampling methods included both snapshot, live sampling of the pelagic zone, and historical approaches, with diapauses eggs collected from sediments.

Studies of zooplankton in South Australia water bodies are not extensive but reveal a wealth of biodiversity. The statistics provided by ABRS under the Australian Faunal Directory based on morphological description includes 33 families in the Phylum Rotifera, 9 families in the Class Branchiopoda and 83 families in Subclass Copepoda till to date. These studies give us a snapshot of the systematic work done previously in Australia with many first records and novel species identified.

The identity of zooplankton in South Australian drinking water reservoirs is still largely unexplored, although preliminary analyses on the structure of zooplankton communities in other areas of Australia have been made see Shiel et al. (2006), Colbourne et al. (2006), Ricci et al. (2003), Shiel et al. (1982) for more detail. Considering the ubiquity and critical role that zooplankton play in the maintenance and health of aquatic freshwater habitats, especially in the context of trophic interactions in reservoirs, understanding the community structure becomes important.

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Our research focuses on studying zooplankton diversity by means of genetic and morphological data, for two South Australian Reservoirs which represent habitat with same Mediterranean climate but different water sources, management and mixing cycles.

To bridge difference between genes and ecosystem and to link molecular and cellular functions to population level responses in nature, genomic tools can play an important role. One such genomic tool is the sequencing of Cytochrome c oxidase I gene (COI), which is one of the protein coding genes found in mitochondrial genomes coding for terminal enzymes of respiratory chains (Stryer 1995). The conserved sequence of 5' region of COI which is 650bp long is used as a platform for universal DNA barcoding, because it possesses a greater range of phylogenetic signal than any other mitochondrial gene and is very robust working due to its maternal inheritance (Frézala, 2008 #434)(Hebert, 2003 #435) and has been found to be successful for studying ecological and evolutionary pattern in various groups like fish (Ward, 2005 #297),bird (Hebert PD, 2004 #438; Kerr KC, 2009 #439), crustaceans (Barrett , 2005 #436)(Costa, 2007 #437), and for marine and freshwater zooplankton species (Belyaeva, 2009 #362; Bucklin, 1994 #383; Bucklina , 2004 #386; Gutierrez, 2008 #326; Havel, 2000 #74; Jr., #408).

The current study provides reliable molecular method for extracting and amplifying DNA from alcohol preserved sample using two commercially available extraction kits.

Sampling Sites:

Myponga Reservoir Constructed in 1962, the reservoir is situated approximately 70 km south of Adelaide. The reservoir has a surface area of 2.8 km2 and catchment area of 124 km2 which holds approx. 26,800 mega litres of water with a maximum depth of 42 m at full supply level (Brookes, Hipsey et al. 2005).Unlike other reservoirs it doesn't receives water from river Murray.

It is a highly managed reservoir which has a history of algal blooms, for which artificial mixing and copper sulphate has been used as the control agent for the algal biomass.

This reservoir receives water from its own catchment area and is thermally destratified due to the artificial mixing that is carried out between October to March annually (Lewis, Elliott et al. 2003).

South Para Reservoir Constructed in 1958, the reservoir is situated approximately 60 km north of Adelaide and is thermally stratified. It has a surface area of 4 km2, and catchment area of 228 km2, which holds approx. 45,330 mega litres of water is second largest reservoir in South Australia (Maisano et al, 2005).

This reservoir is not affected by the use of copper sulphate and receives part of its water from the River Murray as well as from its own catchment.

Material and Methods:

Sampling:

Zooplankton Sampling

Live zooplankton were collected by vertical and horizontal hauls using 43 µm mesh plankton net for the period between August 2008 to June 2010 in mid afternoon on a monthly basis. Samples were collected at different sites ranging from deep, shallow and land-water interface area. The collections were preserved in 70% ethanol till further analysis, 28 species where identified and classified morphologically into Class Branchiopoda, Phylum Rotifera and Subclass Copepoda respectively, for species distribution see Table 1.

Table 1: Zooplankton diversity in Myponga and South Para Reservoir

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Species Name

Myponga

South Para

Cladocera

Daphnia lumholtzi

√

√

Daphnia carinata

√

√

Eubosmina meridionalis

√

√

Ceriodaphnia cornuta

√

√

Diaphanosoma unguiculatum

√

√

Dunhevedia crassa crassa

√

X

Ceriodaphnia sp.A

√

√

Ceriodaphnia sp.B

√

√

Moina sp

√

X

Copepoda

Microcyclops sp A

√

X

Microcyclops sp B

x

√

Boeckella symmetrica

√

√

Boeckella triarticulata

√

√

Tropocyclops sp B

X

√

Calamoecia ampulla

√

√

Australocyclops australis

√

X

Australocyclops similis

x

√

Mesocyclops notius

√

√

Microcyclops varicans

√

√

Rotifera

Asplanchna priodonta

√

√

Keratella cochlearis

x

√

Keratella procurva

√

√

Keratella slacki

√

√

Epiphanes macroura

X

X

Lecane bulla

√

√

Lecane closterocerca

√

X

Lepadella acuminata

√

X

Lepadella patella

X

√

Key: √ = Present; X = Absent

Diapause eggs collection

In sediment most viable eggs occur in the upper few centimeters known as the 'active egg bank' (Caceres and Hairston, 1998) this egg bank is reflective of the reservoirs recent zooplankton assemblage. Alternatively deeper sediment cores help to reconstruct historical zooplankton assemblages that may be linked to historical changes in habitats or colonization processes. Sediment samples were taken from the land water interface using a sediment corer 1meter in length and 40 mm in diameter from strategic points around the reservoirs.

The sediment sample where wrapped in aluminium foil and kept at 4°c till further analysis with sucrose flotation.

Sucrose Flotation Protocol

The sucrose flotation technique has been standardised in our laboratory (Furst, 2008 #378), where 5gm of sediment was placed in a 50 mL sterile falcon tube to which a 1.75 M sucrose solution was added until the contents of the tube weighs 80 g. The sediment was gently mixed to suspend it in the sucrose solution and was placed in a centrifuge at 100g for 15 min.

After centrifugation, particles denser than the sucrose solution settle's to the bottom of the falcon tube while those that are lighter, including diapause eggs, are suspended in the supernatant. The supernatant was then passed through a 47 µm filter and rinsed with distilled water until all traces of the sucrose solution were removed. The collected materials were transferred to a Petri dish with a 10 mL plastic pipette.

The collected diapause eggs were then incubated in the Petri dish under a constant illumination of 10000 Lux and observed daily for hatched neonates.

DNA Extraction Protocol

Two commercially available genomic DNA extraction kit (Chelex® 100 Bio-Rad and QIAamp® DNA mini kit from Qiagen) was used for study. The QIAamp® DNA mini kit from Qiagen was found to be highly successful in extracting DNA from cladocerans and rotifers but limited success with copepod.

The extraction protocol was modified then the procedure described by manufacturer, it involved following steps

Add 50µl of Buffer ATL to 1.5ml microfuge tube containing specimen and put it in shaker for 45sec.

Freeze thaw 7 times at -70°C (using liquid nitrogen) and 56°c alternatively.

Add 20µl of Proteinase K to the tube, place it in shaker for 30sec and incubate at 56°c for 2hrs cover the tube with aluminium foil during incubation.

Add 100 µl of Buffer AL to the sample, place it in shaker for 45sec and incubate it at 70°c for 10min again cover the tube with aluminium foil during incubation.

After incubation, allow the tube to get cool till room temperature and then centrifuge at full speed for 45sec to precipitate any undigested pellet.

Transfer the supernatant into a new 1.5ml tube.

Add 200µlof denatured ethanol to the sample and place it in shaker for 30 sec.

Carefully transfer the mixture to QIAamp spin column without wetting the rim and centrifuge the column at 6000 rcf for 1 min.

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Place the spin column in a new collection tube; discard the tube containing the filtrate.

Add 500µl of Buffer AW1 without wetting the rim and keep it in shaker for 30sec; then centrifuge at 6000rcf for 1min.

Place the spin column in a clean collection tube; discard the tube containing the filtrate.

Add 500µl of Buffer AW2 without wetting the rim and keep it in shaker for 30sec; then centrifuge at full speed for 3 min.

Place spin column into a clean 1.5ml collection tube; discard the tube containing the filtrate.

Add 40µl of pre warmed (50°c) molecular grade water or Buffer AE and incubate the tube at room temperature for 90 sec.

Centrifuge the tube at 6000rpm for 1 min.

Save the filtrate containing DNA and then store the extraction at -20°c.

For copepod especially for Calamoecia ampulla and rotifer the Chelex® 100 Bio-Rad method, hereafter referred to as "Chelex" was found to be highly successful. The organism(s) where placed in 35 μL of Chelex at 4°C overnight and a protocol adapted from Mills,2006 (Mills, 2006 #250) where the tube containing Chelex and specimen was incubated at 56°C for 20 min, 99°C for 10 min, and then cooled at 4°C for 30 min was used. Following this incubation the tube where then centrifuged at 6000 rpm for 1 min to precipitate Chelex beads, DNA was extracted and stored in a new 1.5ml tube at 4°C until needed.

PCR Amplification and Purification Protocol

A 650 base pair segment of COI was amplified with PCR using LCO 1490 and HCO 2198 primers for each individuals. The 25µl PCR reaction mix included 16.8µl of ultrapure water, 2.5µl of 10x PCR Buffer, 1.5µl of 50mM MgCl2, 0.75µl each of 10µM LCO 1490 and HCO 2198, 0.5µl of 10µM dNTP's ,0.12µl of Taq Platinum Polymerase and 2µl of DNA Template. Samples where then subjected to the following thermo cycling profile: 1cycle of 94°C for 3 min, 5 cycles at 94°c for 60sec, 46°c for 60sec and 72°c for 90sec followed by 35 cycles of 94°c for 15sec, 50°c for 15sec and 72°c for 30sec with a final incubation at 72°c for 5 min.PCR product was then visualised in precast Agarose gel and the most intense product where selected for sequencing. The positive band PCR product was purified by using Montage PCR Centrifugal Filter Devices.

DNA Sequencing Protocol

The Capillary Sequencing (CS) method was found to be much more reliable than the Purified DNA (PD) due to low recommended quantity of DNA required for sequencing.

In capillary sequencing method, purified PCR product was sequenced using 1µl of Big Dye Terminator, 3.5µl of Big Dye buffer, 0.5µl of 10µM of each primer and 1-3µl of DNA product (Where 1 =strong band with DNA concentration >8ng/ µl and 3= weak band with DNA concentration <3ng/ µl), the total volume was made up to 25µl using ultrapure water. The sample where then subjected to the following thermo cycling profile : 25 cycle at 96°c for 30sec, 50°c for 15 sec and 60°c for 4min with a final incubation at 25°C. The DNA product was then cleaned up using MultiScreenTMHTS Vacuum manifold. The product was then send to be sequenced bidirectional on AB 3730xl Capillary sequencer.

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